Compositions, kits and related methods for the detection and/or monitoring of listeria

ABSTRACT

Provided are compositions, kits, and methods for the identification of  Listeria.  In certain aspects and embodiments, the compositions, kits, and methods may provide improvements in relation to specificity, sensitivity, and speed of detection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Application 61/141,651,filed Dec. 30, 2008 and is hereby incorporated by reference in itsentirety.

FIELD

The inventions disclosed herein relate to microorganisms and tocompositions and mechanisms for the detection thereof.

BACKGROUND

This application relates to compositions and methods for detectingListeria. In certain aspects and embodiments, particular regions of theListeria 16S rRNA have been identified as preferred targets for nucleicacid amplification reactions.

Listeria is a gram-positive bacteria found in soil and water. Vegetablescan become contaminated from the soil or from manure used as fertilizer.Animals can carry the bacterium without appearing ill and cancontaminate foods of animal origin such as meats and dairy products.Listeriosis, a serious infection caused by eating food contaminated withthe bacterium Listeria monocytogenes, has recently been recognized as animportant public health problem in the United States. In the UnitedStates, an estimated 2,500 persons become seriously ill with listeriosiseach year. Of these, 500 die. At increased risk are pregnant women,newborns, persons with weakened immune systems; persons with cancer,diabetes, or kidney disease; persons with AIDS, persons who takeglucocorticosteroid medications, and the elderly. Species include L.monocytogenes, L. innocua, L. welshimeri, L. ivanovii, L. seeligeri, L.grayi, and L. murrayi.

SUMMARY

The present invention relates to compositions, kits, and methods used inthe detection of Listeria. The invention is based in part on thediscovery that certain Listeria sequences are surprisingly efficaciousfor the detection of Listeria. In certain aspects and embodiments,particular regions of the Listeria 16S rRNA have been identified aspreferred targets for nucleic acid amplification reactions which provideimprovements in relation to specificity, sensitivity, or speed ofdetection as well as other advantages.

In some preferred aspects, there are provided compositions for use in aListeria transcription-mediated amplification assay (hereinafter “TMA”).In some preferred aspects, there are provided kits for performing aListeria transcription-mediated amplification assay. In some preferredaspects, there are provided methods for performing a Listeriatranscription-mediated amplification assay. In certain preferredembodiments, the compositions, kits, and/or methods may include or useone or more oligonucleotides such as a: T7 provider oligonucleotide,primer oligonucleotide, detection oligonucleotide, blockeroligonucleotide, Torch oligonucleotide, and the like.

Therefore, according to one aspect, there are provided compositions foruse in a Listeria nucleic acid amplification assay. In certain preferredembodiments, the compositions include a T7 provider oligonucleotide anda primer oligonucleotide; in which the T7 provider oligonucleotidetargets a sequence in a region of Listeria nucleic acid corresponding tonucleotide positions of about 364-440 of E. coli 16S rRNA and the primeroligonucleotide targets a sequence in a region of Listeria nucleic acid,in which the T7 provider oligonucleotide and primer oligonucleotide usedin the amplification assay target opposite strands of the Listerianucleic acid sequence to be amplified.

In a second aspect, there are provided compositions for use in aListeria nucleic acid amplification assay. In certain preferredembodiments, the compositions include a first T7 provideroligonucleotide having the sequence of SEQ ID NO: 13 or complement and asecond T7 provider oligonucleotide having the sequence of SEQ ID NO: 14or complement. In another preferred embodiment, the primeroligonucleotide has the sequence of SEQ ID NO: 23 or complement.

In a third aspect, there are provided compositions for use in a Listerianucleic acid amplification assay. In certain preferred embodiments, thecompositions include two or more T7 provider oligonucleotides and one ormore primer oligonucleotides. In certain preferred embodiments, the twoor more T7 provider oligonucleotides and the one or more primeroligonucleotides are configured and arranged such that L. monocytogenes,L. innocua, L. grayi, L. ivanovii, L. welshimeri, L. murrayi, and L.seeligeri, are amplified under the Listeria nucleic acid amplificationassay conditions. In certain preferred embodiments, the two or more T7provider oligonucleotides and the one or more primer oligonucleotidesare configured and arranged such that Brochothrix thermosphacta andErysipelothrix rhusiopathiae are not substantially amplified under theListeria nucleic acid amplification assay conditions.

In a fourth aspect, there are provided kits that include thecompositions provided herein. In certain preferred embodiments of theaspects provided herein, the kits include a T7 provider oligonucleotideand a primer oligonucleotide, in which the T7 provider oligonucleotidetargets a sequence in a region of Listeria nucleic acid corresponding tonucleotide positions of about 364-440 of E. coli 16S rRNA and the primeroligonucleotide targets a sequence in a region of Listeria nucleic acid,in which the T7 provider oligonucleotide and primer oligonucleotide usedin the amplification assay target opposite strands of the Listerianucleic acid sequence to be amplified.

In a fifth aspect, there are provided kits that include the compositionsprovided herein. In certain preferred embodiments of the aspectsprovided herein, the kits include a first T7 provider oligonucleotidehaving the sequence of SEQ ID NO: 13 or complement and a second T7provider oligonucleotide having the sequence of SEQ ID NO: 14 orcomplement. In another preferred embodiment, the primer oligonucleotidehas the sequence of SEQ ID NO: 23 or complement.

In a sixth aspect, there are provided kits that include the compositionsprovided herein. In certain preferred embodiments of the aspectsprovided herein, the kits include two or more T7 provideroligonucleotides and one or more primer oligonucleotides. In certainpreferred embodiments, the two or more T7 provider oligonucleotides andthe one or more primer oligonucleotides are configured and arranged suchthat L. monocytogenes, L. innocua, L. grayi, L. ivanovii, L. welshimeri,L. murrayi, and L. seeligeri, are amplified under the Listeria nucleicacid amplification assay conditions. In certain preferred embodiments,the two or more T7 provider oligonucleotides and the one or more primeroligonucleotides are configured and arranged such that Brochothrixthermosphacta and Erysipelothrix rhusiopathiae are not substantiallyamplified under the Listeria nucleic acid amplification assayconditions.

In a seventh aspect, there are provided methods for detecting thepresence of Listeria in a sample using the compositions and/or kitsprovided herein. In certain preferred embodiments of the aspectsprovided herein, the methods use a T7 provider oligonucleotide and aprimer oligonucleotide, in which the T7 provider oligonucleotide targetsa sequence in a region of Listeria nucleic acid corresponding tonucleotide positions of about 364-440 of E. coli 16S rRNA and the primeroligonucleotide targets a sequence in a region of Listeria nucleic acid,in which the T7 provider oligonucleotide and primer oligonucleotide usedin the amplification assay target opposite strands of the Listerianucleic acid sequence to be amplified.

In an eighth aspect, there are provided methods for detecting thepresence of Listeria in a sample using the compositions and/or kitsprovided herein. In certain preferred embodiments of the aspectsprovided herein, the methods use a first T7 provider oligonucleotidehaving the sequence of SEQ ID NO: 13 or complement and a second T7provider oligonucleotide having the sequence of SEQ ID NO: 14 orcomplement. In another preferred embodiment, the primer oligonucleotidehas the sequence of SEQ ID NO: 23 or complement.

In a ninth aspect, there are provided methods for detecting the presenceof Listeria in a sample using the compositions and/or kits providedherein. In certain preferred embodiments of the aspects provided herein,the methods use two or more T7 provider oligonucleotides and one or moreprimer oligonucleotides. In certain preferred embodiments, the two ormore T7 provider oligonucleotides and the one or more primeroligonucleotides are configured and arranged such that L. monocytogenes,L. innocua, L. grayi, L. ivanovii, L. welshimeri, L. murrayi, and L.seeligeri, are amplified under the Listeria nucleic acid amplificationassay conditions. In certain preferred embodiments, the two or more T7provider oligonucleotides and the one or more primer oligonucleotidesare configured and arranged such that Brochothrix thermosphacta andErysipelothrix rhusiopathiae are not substantially amplified under theListeria nucleic acid amplification assay conditions.

In one particularly preferred embodiment of the aspects provided herein,the T7 provider oligonucleotide has an adenine at the nucleotideposition that is complementary to the nucleotide position in a Listerianucleic acid sequence corresponding to nucleotide position 407 of E.coli 16S rRNA. In another particularly preferred embodiment of theaspects provided herein, the T7 provider oligonucleotide has a guanineat the nucleotide position that is complementary to the nucleotideposition in a Listeria nucleic acid sequence corresponding to nucleotideposition 407 of E. coli 16S rRNA. In one preferred embodiment, the T7provider oligonucleotide has a sequence selected from the sequences ofSEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 15, or complements. In aparticularly preferred embodiment of the aspects provided herein, the T7provider oligonucleotide targets a sequence in a region of Listerianucleic acid corresponding to nucleotide positions of about 398-417 ofE. coli 16S rRNA. In one particularly preferred embodiment of theaspects provided herein, the T7 provider oligonucleotides comprises thesequence of SEQ ID NO: 13 or complement. In another particularlypreferred embodiment of the aspects provided herein, the T7 provideroligonucleotides comprises the sequence of SEQ ID NO: 14 or complement.

In another particularly preferred embodiment of the aspects providedherein, the composition further comprises a second T7 provider. In oneparticularly preferred embodiment of the aspects provided herein, thecomposition includes a first T7 provider oligonucleotide having anadenine at the nucleotide position that is complementary to thenucleotide position in a Listeria nucleic acid sequence corresponding tonucleotide position 407 of E. coli 16S rRNA, and a second T7 provideroligonucleotide having a guanine at the nucleotide position that iscomplementary to the nucleotide position in a Listeria nucleic acidsequence corresponding to nucleotide position 407 of E. coli 16S rRNA.In another preferred embodiment, at least one of the T7 provideroligonucleotides has a sequence selected from the sequences of SEQ IDNOs: 8, 9, 10, 11, 12, 13, 14, 15, or complements. In one preferredembodiment of the aspects provided herein, at least one of the T7provider oligonucleotides targets a sequence in a region of Listerianucleic acid corresponding to nucleotide positions of about 398-417 ofE. coli 16S rRNA. In another particularly preferred embodiment of theaspects provided herein in which the composition includes a first and asecond T7 provider oligonucleotide, the first and the second T7 provideroligonucleotide each target a sequence in a region of Listeria nucleicacid corresponding to nucleotide positions of about 398-417 of E. coli16S rRNA. In other particularly preferred embodiments of the aspectsprovided herein in which the composition includes a first and a secondT7 provider oligonucleotide, the first T7 provider oligonucleotidescomprises the sequence of SEQ ID NO: 13 or complement, and the second T7provider oligonucleotides comprises the sequence of SEQ ID NO: 14 orcomplement.

In one preferred embodiment of the aspects provided herein, the primeroligonucleotide targets a sequence in a region of Listeria nucleic acidcorresponding to nucleotide positions of about 439-505 of E. coli 16SrRNA. In a preferred embodiment, the primer oligonucleotide targets asequence in a region of Listeria nucleic acid corresponding tonucleotide positions of about 480-501 of E. coli 16S rRNA. In yetanother preferred embodiment, the primer oligonucleotide has a sequenceselected from the sequences of SEQ ID NOs: 16, 17, 18, 19, 20, 21, 22,23, or complements. In a particularly preferred embodiment, the primeroligonucleotide comprises the sequence of SEQ ID NO: 23 or complement.

In certain preferred embodiments of the aspects provided herein, the T7provider oligonucleotide includes 15-35 nucleotides that are at least70%; or 75%; or 80%; or 85%; or 90%; or 100% complementary to thetargeted Listeria nucleic acid sequence. In certain preferredembodiments, the T7 provider oligonucleotide includes 15-35 nucleotidesthat are complementary to the targeted Listeria nucleic acid sequencebut have 1 mismatch; or 2 mismatches; or 3 mismatches; or 4 mismatches,or 5 mismatches as compared to the targeted nucleic acid sequence withinthe 15-35 complementary nucleotides.

In some preferred embodiments of the aspects provided herein, one ormore additional oligonucleotide types and/or other amplificationreagents that serve to facilitate or improve one or more aspects of thetranscription-mediated amplification reaction may be included. Forexample, in a preferred embodiment, in addition to a T7 provideroligonucleotide and/or a primer oligonucleotide, additionaloligonucleotides may further include one or more of a: detectionoligonucleotide, blocker oligonucleotide, target captureoligonucleotide, helper oligonucleotide, and the like.

In some preferred embodiments of the aspects provided herein, thecompositions, kits, and/or methods may further include or use adetection oligonucleotide, preferably a torch oligonucleotide or anacridinium ester probe. In one preferred embodiment, the detectionoligonucleotide is a torch oligonucleotide selected from the sequencesof SEQ ID NOs: 24-28 or complements. In a particularly preferredembodiment, the detection oligonucleotide is a torch oligonucleotidehaving the sequence of SEQ ID NO: 27 or complement.

In some preferred embodiments of the aspects provided herein, thecompositions, kits, and/or methods may further include or use a blockeroligonucleotide. In one preferred embodiment, the blockeroligonucleotide is selected from the sequences of SEQ ID NOs: 1-7 orcomplements. In a particularly preferred embodiment, the blockeroligonucleotide has the sequence of SEQ ID NO: 6 or complement.

In some preferred embodiments of the aspects provided herein, thecompositions, kits, and/or methods may further include or use a targetcapture oligonucleotide. In one preferred embodiment, the target captureoligonucleotide is selected from the sequences of SEQ ID NOs: 29-36 orcomplements. In a particularly preferred embodiment, the target captureoligonucleotide has the sequence of SEQ ID NOs: 31 or complement.

In some preferred embodiments of the aspects provided herein, thecompositions, kits, and/or methods may further include or use a helperoligonucleotide.

The terms and concepts of the invention have meanings as set forthherein unless expressly stated to the contrary and/or unless contextspecifically dictates otherwise. Unless defined otherwise, scientificand technical terms used herein have the same meaning as commonlyunderstood by those skilled in the relevant art. General definitions maybe found in technical books relevant to the art of molecular biology,e.g., Dictionary of Microbiology and Molecular Biology, 2nd ed.(Singleton et al., 1994, John Wiley & Sons, New York, N.Y.) or TheHarper Collins Dictionary of Biology (Hale & Marham, 1991, HarperPerennial, New York, N.Y.). Unless mentioned otherwise, techniquesemployed or contemplated herein are standard methodologies well known toone of ordinary skill in the art. The examples included hereinillustrate some preferred embodiments. Each reference cited herein isspecifically incorporated herein by reference in its entirety.

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, “a nucleic acid,” is understood torepresent one or more nucleic acids. As such, the terms “a” (or “an”),“one or more,” and “at least one” can be used interchangeably herein.

The term “nucleic acid” as used herein encompasses a singular “nucleicacid” as well as plural “nucleic acids,” and refers to any chain of twoor more nucleotides, nucleosides, or nucleobases (e.g.,deoxyribonucleotides or ribonucleotides) covalently bonded together.Nucleic acids include, but are not limited to, virus genomes, orportions thereof, either DNA or RNA, bacterial genomes, or portionsthereof, fungal, plant or animal genomes, or portions thereof, messengerRNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), plasmid DNA,mitochondrial DNA, or synthetic DNA or RNA. A nucleic acid may beprovided in a linear (e.g., mRNA), circular (e.g., plasmid), or branchedform, as well as a double-stranded or single-stranded form. Nucleicacids may include modified bases to alter the function or behavior ofthe nucleic acid, e.g., addition of a 3′-terminal dideoxynucleotide toblock additional nucleotides from being added to the nucleic acid. Asused herein, a “sequence” of a nucleic acid refers to the sequence ofbases which make up a nucleic acid.

The term “polynucleotide” as used herein denotes a nucleic acid chain.Throughout this application, nucleic acids are designated by the5′-terminus to the 3′-terminus. Standard nucleic acids, e.g., DNA andRNA, are typically synthesized “3′-to-5′,” i.e., by the addition ofnucleotides to the 5′-terminus of a growing nucleic acid.

A “nucleotide” as used herein is a subunit of a nucleic acid consistingof a phosphate group, a 5-carbon sugar and a nitrogenous base. The5-carbon sugar found in RNA is ribose. In DNA, the 5-carbon sugar is2′-deoxyribose. The term also includes analogs of such subunits, such asa methoxy group at the 2′ position of the ribose (2′-O-Me). As usedherein, methoxy oligonucleotides containing “T” residues have a methoxygroup at the 2′ position of the ribose moiety, and a uracil at the baseposition of the nucleotide.

A “non-nucleotide unit” as used herein is a unit which does notsignificantly participate in hybridization of a polymer. Such units mustnot, for example, participate in any significant hydrogen bonding with anucleotide, and would exclude units having as a component one of thefive nucleotide bases or analogs thereof.

A “target nucleic acid” as used herein is a nucleic acid comprising a“target sequence” to be amplified. Target nucleic acids may be DNA orRNA as described herein, and may be either single-stranded ordouble-stranded. The target nucleic acid may include other sequencesbesides the target sequence which may not be amplified. Typical targetnucleic acids include virus genomes, bacterial genomes, fungal genomes,plant genomes, animal genomes, rRNA, tRNA, or mRNA from viruses,bacteria or eukaryotic cells, mitochondrial DNA, or chromosomal DNA.

By “isolated” it is meant that a sample containing a target nucleic acidis taken from its natural milieu, but the term does not connote anydegree of purification.

The term “target sequence” as used herein refers to the particularnucleotide sequence of the target nucleic acid which is to be amplified.The “target sequence” includes the complexing sequences to whicholigonucleotides (e.g., priming oligonucleotides and/or promoteroligonucleotides) complex during the processes of TMA. Where the targetnucleic acid is originally single-stranded, the term “target sequence”will also refer to the sequence complementary to the “target sequence”as present in the target nucleic acid. Where the “target nucleic acid”is originally double-stranded, the term “target sequence” refers to boththe sense (+) and antisense (−) strands. In choosing a target sequence,the skilled artisan will understand that a “unique” sequence should bechosen so as to distinguish between unrelated or closely related targetnucleic acids.

The term “targets a sequence” as used herein in reference to a region ofListeria nucleic acid refers to a process whereby an oligonucleotidehybridizes to the target sequence in a manner that allows foramplification and detection as described herein. In one preferredembodiment, the oligonucleotide is complementary with the targetedListeria nucleic acid sequence and contains no mismatches. In anotherpreferred embodiment, the oligonucleotide is complementary but contains1; or 2; or 3; or 4; or 5 mismatches with the targeted Listeria nucleicacid sequence. Preferably, the oligonucleotide that hybridizes to theListeria nucleic acid sequence includes at least 10 to 50; or 12 to 45;or 14 to 40; or 15-35 nucleotides complementary to the target sequence.

The term “fragment” or “region” as used herein in reference to theListeria targeted nucleic acid sequence refers to a piece of contiguousnucleic acid. In certain embodiments, the fragment includes 25; or 50;or 75; or 100; or 125; or 150; or 175; or 200; or 225; or 250; or 300;or 350; or 400; or 450; or 500; or 750; or 1000; or 2000; or 3000nucleotides.

As used herein, the term “oligonucleotide” or “oligo” or “oligomer” isintended to encompass a singular “oligonucleotide” as well as plural“oligonucleotides,” and refers to any polymer of two or more ofnucleotides, nucleosides, nucleobases or related compounds used as areagent in the amplification methods disclosed herein, as well assubsequent detection methods. The oligonucleotide may be DNA and/or RNAand/or analogs thereof. The term oligonucleotide does not denote anyparticular function to the reagent, rather, it is used generically tocover all such reagents described herein. An oligonucleotide may servevarious different functions, e.g., it may function as a primer if it isspecific for and capable of hybridizing to a complementary strand andcan further be extended in the presence of a nucleic acid polymerase, itmay provide a promoter if it contains a sequence recognized by an RNApolymerase and allows for transcription (e.g., a T7 Provider), and itmay function to prevent hybridization or impede primer extension ifappropriately situated and/or modified.

As used herein, an oligonucleotide having a nucleic acid sequence“comprising” or “consisting of” or “consisting essentially of” asequence selected from a group of specific sequences means that theoligonucleotide, as a basic and novel characteristic, is capable ofstably hybridizing to a nucleic acid having the exact complement of oneof the listed nucleic acid sequences of the group under stringenthybridization conditions. An exact complement includes the correspondingDNA or RNA sequence.

As used herein, an oligonucleotide “substantially corresponding to” aspecified nucleic acid sequence means that the referred tooligonucleotide is sufficiently similar to the reference nucleic acidsequence such that the oligonucleotide has similar hybridizationproperties to the reference nucleic acid sequence in that it wouldhybridize with the same target nucleic acid sequence under stringenthybridization conditions. One skilled in the art will understand that“substantially corresponding oligonucleotides” can vary from thereferred to sequence and still hybridize to the same target nucleic acidsequence. This variation from the nucleic acid may be stated in terms ofa percentage of identical bases within the sequence or the percentage ofperfectly complementary bases between the probe or primer and its targetsequence. Thus, an oligonucleotide “substantially corresponds” to areference nucleic acid sequence if these percentages of base identity orcomplementarity are from 100% to about 80%. In preferred embodiments,the percentage is from 100% to about 85%. In more preferred embodiments,this percentage can be from 100% to about 90%; in other preferredembodiments, this percentage is from 100% to about 95%. One skilled inthe art will understand the various modifications to the hybridizationconditions that might be required at various percentages ofcomplementarity to allow hybridization to a specific target sequencewithout causing an unacceptable level of non-specific hybridization.

A “helper oligonucleotide” or “helper” refers to an oligonucleotidedesigned to bind to a target nucleic acid and impose a differentsecondary and/or tertiary structure on the target to increase the rateand extent of hybridization of a detection probe or otheroligonucleotide with the targeted nucleic acid, as described, forexample, in U.S. Pat. No. 5,030,557, the contents of which areincorporated by reference herein. Helpers may also be used to assistwith the hybridization to target nucleic acid sequences and function ofprimer, target capture and other oligonucleotides.

As used herein, a “blocking moiety” is a substance used to “block” the3′-terminus of an oligonucleotide or other nucleic acid so that itcannot be efficiently extended by a nucleic acid polymerase.

As used herein, a “priming oligonucleotide” or “primer” is anoligonucleotide, at least the 3′-end of which is complementary to anucleic acid template, and which complexes (by hydrogen bonding orhybridization) with the template to give a primer: template complexsuitable for initiation of synthesis by an RNA- or DNA-dependent DNApolymerase.

As used herein, a “promoter” is a specific nucleic acid sequence that isrecognized by a DNA-dependent RNA polymerase (“transcriptase”) as asignal to bind to the nucleic acid and begin the transcription of RNA ata specific site.

As used herein, a “promoter-provider” or “provider” refers to anoligonucleotide comprising first and second regions, and which ismodified to prevent the initiation of DNA synthesis from its3′-terminus. The “first region” of a promoter-provider oligonucleotidecomprises a base sequence which hybridizes to a DNA template, where thehybridizing sequence is situated 3′, but not necessarily adjacent to, apromoter region. The hybridizing portion of a promoter oligonucleotideis typically at least 10 nucleotides in length, and may extend up to 15,20, 25, 30, 35, 40, 50 or more nucleotides in length. The “secondregion” comprises a promoter sequence for an RNA polymerase. A promoteroligonucleotide is engineered so that it is incapable of being extendedby an RNA- or DNA-dependent DNA polymerase, e.g., reverse transcriptase,preferably comprising a blocking moiety at its 3′-terminus as describedabove. As referred to herein, a “T7 provider” is a blockedpromoter-provider oligonucleotide that provides an oligonucleotidesequence that is recognized by T7 RNA polymerase.

As used herein, a “terminating oligonucleotide” or “blockeroligonucleotide” is an oligonucleotide comprising a base sequence thatis complementary to a region of the target nucleic acid in the vicinityof the 5′-end of the target sequence, so as to “terminate” primerextension of a nascent nucleic acid that includes a primingoligonucleotide, thereby providing a defined 3′-end for the nascentnucleic acid strand.

An “extender oligonucleotide” or “extend oligo” as used herein refers toan oligonucleotide that is the same sense as the T7 Provider and may actas a helper oligonucleotide that opens up structure or improvesspecificity.

As used herein, a “detection oligonucleotide” refers to a nucleic acidoligonucleotide that hybridizes specifically to a target sequence,including an amplified sequence, under conditions that promote nucleicacid hybridization, for detection of the target nucleic acid. By “probeoligonucleotide” or “detection probe” is meant a molecule comprising anoligonucleotide having a base sequence partly or completelycomplementary to a region of a target sequence sought to be detected, soas to hybridize thereto under stringent hybridization conditions.

By “stable” or “stable for detection” is meant that the temperature of areaction mixture is at least 2° C. below the melting temperature of anucleic acid duplex.

By “amplification” or “nucleic acid amplification” is meant productionof multiple copies of a target nucleic acid that contains at least aportion of the intended specific target nucleic acid sequence, asfurther described herein. The multiple copies may be referred to asamplicons or amplification products.

The term “amplicon” as used herein refers to the nucleic acid moleculegenerated during an amplification procedure that is complementary orhomologous to a sequence contained within the target sequence.

By “preferentially hybridize” is meant that under stringenthybridization assay conditions, probes hybridize to their targetsequences, or replicates thereof, to form stable probe: target hybrids,while at the same time formation of stable probe: non-target hybrids isminimized. Thus, a probe hybridizes to a target sequence or replicatethereof to a sufficiently greater extent than to a non-target sequence,to enable one having ordinary skill in the art to accurately quantitatethe RNA replicates or complementary DNA (cDNA) of the target sequenceformed during the amplification.

“Specific hybridization” is an indication that two nucleic acidsequences share a high degree of complementarity. Specific hybridizationcomplexes form under permissive annealing conditions and remainhybridized after any subsequent washing steps. Permissive conditions forannealing of nucleic acid sequences are routinely determinable by one ofordinary skill in the art and may occur, for example, at 65° C. in thepresence of about 6×SSC. Stringency of hybridization may be expressed,in part, with reference to the temperature under which the wash stepsare carried out. Such temperatures are typically selected to be about 5°C. to 20° C. lower than the thermal melting point (Tm) for the specificsequence at a defined ionic strength and pH. The Tm is the temperature(under defined ionic strength and pH) at which 50% of the targetsequence hybridizes to a perfectly matched probe. Equations forcalculating Tm and conditions for nucleic acid hybridization are knownin the art.

By “complementary” is meant that the nucleotide sequences of similarregions of two single-stranded nucleic acids, or to different regions ofthe same single-stranded nucleic acid have a nucleotide base compositionthat allow the single-stranded regions to hybridize together in a stabledouble-stranded hydrogen-bonded region under stringent hybridization oramplification conditions. When a contiguous sequence of nucleotides ofone single-stranded region is able to form a series of “canonical”hydrogen-bonded base pairs with an analogous sequence of nucleotides ofthe other single-stranded region, such that A is paired with U or T andC is paired with G, the nucleotides sequences are “perfectly”complementary.

By “nucleic acid hybrid” or “hybrid” or “duplex” is meant a nucleic acidstructure containing a double-stranded, hydrogen-bonded region whereineach strand is complementary to the other, and wherein the region issufficiently stable under stringent hybridization conditions to bedetected by means including, but not limited to, chemiluminescent orfluorescent light detection, autoradiography, or gel electrophoresis.Such hybrids may comprise RNA:RNA, RNA:DNA, or DNA:DNA duplex molecules.

As used herein, a “target capture oligonucleotide” refers to a nucleicacid oligomer that specifically hybridizes to a target sequence in atarget nucleic acid by standard base pairing and joins to a bindingpartner on an immobilized probe to capture the target nucleic acid to asupport. One example of a target capture oligomer includes two bindingregions: a sequence-binding region (i.e., target-specific portion) andan immobilized probe-binding region, usually on the same oligomer,although the two regions may be present on two different oligomersjoined together by one or more linkers.

As used herein, an “immobilized oligonucleotide”, “immobilized probe” or“immobilized nucleic acid” refers to a nucleic acid binding partner thatjoins a target capture oligomer to a support, directly or indirectly. Animmobilized oligonucleotide joined to a support facilitates separationof a target capture bound target from unbound material in a sample.

As used herein, a “label” refers to a moiety or compound joined directlyor indirectly to a probe that is detected or leads to a detectablesignal.

As used herein, structures referred to as “molecular torches” aredesigned to include distinct regions of self-complementarity (coined“the target binding domain” and “the target closing domain”) which areconnected by a joining region and which hybridize to one another underpredetermined hybridization assay conditions.

As used herein, a “DNA-dependent DNA polymerase” is an enzyme thatsynthesizes a complementary DNA copy from a DNA template. Examples areDNA polymerase I from E. coli, bacteriophage T7 DNA polymerase, or DNApolymerases from bacteriophages T4, Phi-29, M2, or T5. DNA-dependent DNApolymerases may be the naturally occurring enzymes isolated frombacteria or bacteriophages or expressed recombinantly, or may bemodified or “evolved” forms which have been engineered to possesscertain desirable characteristics, e.g., thermostability, or the abilityto recognize or synthesize a DNA strand from various modified templates.All known DNA-dependent DNA polymerases require a complementary primerto initiate synthesis. It is known that under suitable conditions aDNA-dependent DNA polymerase may synthesize a complementary DNA copyfrom an RNA template. RNA-dependent DNA polymerases typically also haveDNA-dependent DNA polymerase activity.

As used herein, a “DNA-dependent RNA polymerase” or “transcriptase” isan enzyme that synthesizes multiple RNA copies from a double-stranded orpartially-double-stranded DNA molecule having a promoter sequence thatis usually double-stranded. The RNA molecules (“transcripts”) aresynthesized in the 5′-to-3′ direction beginning at a specific positionjust downstream of the promoter. Examples of transcriptases are theDNA-dependent RNA polymerase from E. coli and bacteriophages T7, T3, andSP6.

As used herein, an “RNA-dependent DNA polymerase” or “reversetranscriptase” (“RT”) is an enzyme that synthesizes a complementary DNAcopy from an RNA template. All known reverse transcriptases also havethe ability to make a complementary DNA copy from a DNA template; thus,they are both RNA- and DNA-dependent DNA polymerases. RTs may also havean RNAse H activity. A primer is required to initiate synthesis withboth RNA and DNA templates.

As used herein, a “selective RNAse” is an enzyme that degrades the RNAportion of an RNA:DNA duplex but not single-stranded RNA,double-stranded RNA or DNA. An exemplary selective RNAse is RNAse H.Enzymes other than RNAse H which possess the same or similar activitymay also be used. Selective RNAses may be endonucleases or exonucleases.Most reverse transcriptase enzymes contain an RNAse H activity inaddition to their polymerase activities. However, other sources of theRNAse H are available without an associated polymerase activity. Thedegradation may result in separation of RNA from a RNA:DNA complex.Alternatively, a selective RNAse may simply cut the RNA at variouslocations such that portions of the RNA melt off or permit enzymes tounwind portions of the RNA. Other enzymes which selectively degrade RNAtarget sequences or RNA products of the present invention will bereadily apparent to those of ordinary skill in the art.

The term “specificity,” in the context of an amplification system, isused herein to refer to the characteristic of an amplification systemwhich describes its ability to distinguish between target and non-targetsequences dependent on sequence and assay conditions. In terms of anucleic acid amplification, specificity generally refers to the ratio ofthe number of specific amplicons produced to the number of side-products(i.e., the signal-to-noise ratio).

The term “sensitivity” is used herein to refer to the precision withwhich a nucleic acid amplification reaction can be detected orquantitated. The sensitivity of an amplification reaction is generally ameasure of the smallest copy number of the target nucleic acid that canbe reliably detected in the amplification system, and will depend, forexample, on the detection assay being employed, and the specificity ofthe amplification reaction, i.e., the ratio of specific amplicons toside-products.

As used herein, a “colony forming unit” (“CFU”) is used as a measure ofviable microorganisms in a sample. A CFU is an individual viable cellcapable of forming on a solid medium a visible colony whose individualcells are derived by cell division from one parental cell. One CFUcorresponds to ˜1000 copies of rRNA.

As used herein, the term “TTime” is the threshold time or time ofemergence of signal in a real-time plot of the assay data. TTime valuesestimate the time at which a particular threshold indicating ampliconproduction is passed in a real-time amplification reaction. TTime and analgorithm for calculating and using TTime values are described in Lightet al., U.S. Pub. No. 2006/0276972, paragraphs [0517] through [0538],the disclosure of which is hereby incorporated by reference herein. Acurve fitting procedure is applied to normalized and background-adjusteddata. The curve fit is performed for only a portion of the data betweena predetermined low bound and high bound. The goal, after finding thecurve that fits the data, is to estimate the time corresponding to thepoint at which the curve or a projection thereof intersects a predefinedthreshold value. In one embodiment, the threshold for normalized data is0.11. The high and low bounds are determined empirically as that rangeover which curves fit to a variety of control data sets exhibit theleast variability in the time associated with the given threshold value.For example, in one embodiment, the low bound is 0.04 and the high boundis 0.36. The curve is fit for data extending from the first data pointbelow the low bound through the first data point past the high bound.Next, there is made a determination whether the slope of the fit isstatistically significant. For example, if the p value of the firstorder coefficient is less than 0.05, the fit is considered significant,and processing continues. If not, processing stops. Alternatively, thevalidity of the data can be determined by the R² value. The slope m andintercept b of the linear curve y=mx+b are determined for the fittedcurve. With that information, TTime can be determined using thefollowing equation:

TTime=(Threshold−b)/m

As used herein, the term “relative fluorescence unit” (“RFU”) is anarbitrary unit of measurement of fluorescence intensity. RFU varies withthe characteristics of the detection means used for the measurement.

As used herein, the term “real-time TMA” refers to single-primertranscription-mediated amplification (“TMA”) of target nucleic acid thatis monitored by real-time detection means.

The term “about” as used herein in the context of nucleotide positionsmeans the indicated position ±1 nucleotide; or ±2 nucleotides; or ±3nucleotides; or ±4 nucleotides; or ±5 nucleotides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C show generic examples of amplification charts for analyteshowing (1A) “Poor”, (1B) “Better”, and (1C) “Good” assay performance.

FIG. 2 shows sequences encompassing the Listeria “450” amplification anddetection region (corresponding to bp 350-505 of the 16S rRNA of E.coli, Accession No. J01859).

FIG. 3 shows sequences encompassing the Listeria “1275” amplificationand detection region (corresponding to bp 1180-1370 of the 16S rRNA ofE. coli, Accession No. J01859).

DETAILED DESCRIPTION

In certain aspects and embodiments, the invention relates tocompositions, methods and kits for the identification, detection, and/orquantitation of Listeria, which may be present either alone or as acomponent, large or small, of a homogeneous or heterogeneous mixture ofnucleic acids in a sample taken for testing, e.g., for diagnostictesting, for screening of blood products, for microbiological detectionin bioprocesses, food, water, industrial or environmental samples, andfor other purposes. Specific methods, compositions, and kits asdisclosed herein provide improved sensitivity, specificity, or speed ofdetection in the amplification-based detection of Listeria. Listeriaribosomal RNA is very closely related to rRNA of Brochothrixthermosphacta and Erysipelothrix rhusiopathiae. Accordingly, in certainembodiments of the invention, the Listeria assay identifies rRNAsequences common to nearly all species, subspecies and serovars of theListeria genus, and differentiates Listeria from other closely relatedspecies. A useful region for such differentiation is the 450 region ofthe 16S rRNA. An alternative region for such differentiation is the 1275region of the 16S rRNA.

As a result of extensive analyses of amplification oligonucleotidesspecific for Listeria, the particular region of Listeria correspondingto the region from about 350 to about 505 bp of E. coli (accession no.J01859) 16S rRNA reference sequence, hereinafter referred to as the “450region”, has been identified as a preferred target foramplification-based detection of Listeria. Accordingly, the inventionrelates to methods of detection of Listeria in a sample of interest,amplification oligonucleotides, compositions, reactions mixtures, kits,and the like.

The Listeria genus assay detects ribosomal RNA sequences specific forknown Listeria species. It utilizes real-time TMA technology, where thetarget-specific sequence is amplified using reverse TMA and a probe isused to detect the amplified products as they are produced. Targetdetection is performed simultaneously with the amplification anddetection of an internal control in order to confirm reliability of theresult. The result of the assay consists of the classification of thesample as positive or negative for the presence or absence of Listeria.The assay is capable of amplifying more than one Listeria species. Insome preferred embodiments, two or more Listeria species selected fromthe group consisting of L. monocytogenes, L. innocua, L. welshimeri, L.ivanovii, L. seeligeri, L. grayi, and L. murrayi are amplified. In otherpreferred embodiments, all of the species are amplified.

In one embodiment, the sample is a biopharmaceutical process(bioprocess) stream where Listeria is a known or suspected contaminant.A “bioprocess,” as used herein, refers generally to any process in whichliving cells or organisms, or components thereof, are present, eitherintended or unintended. For example, essentially any manufacturing orother process that employs one or more samples or sample streams, atleast one of which contains living cells, organisms, or componentsthereof, or contains such cells, organisms or components as a result ofunintended contamination, is considered a bioprocess. In many suchprocesses it is desirable to have the ability to detect, identify and/orcontrol the presence and/or sources of living cells, organisms orcomponents thereof within a process. Using the methods disclosed herein,for example, the presence and/or sources of Listeria in one or morebioprocess samples and/or streams may be monitored in a rapid andsensitive fashion.

Target Nucleic Acid/Target Sequence

Target nucleic acids may be isolated from any number of sources based onthe purpose of the amplification assay being carried out. Sources oftarget nucleic acids include, but are not limited to, clinicalspecimens, e.g., blood, urine, saliva, feces, semen, or spinal fluid,from criminal evidence, from environmental samples, e.g., water or soilsamples, from food, from industrial samples, from cDNA libraries, orfrom total cellular RNA. If necessary, target nucleic acids are madeavailable for interaction with various oligonucleotides. This mayinclude, for example, cell lysis or cell permeabilization to release thetarget nucleic acid from cells, which then may be followed by one ormore purification steps, such as a series of isolation and wash steps.See, e.g., Clark et al., “Method for Extracting Nucleic Acids from aWide Range of Organisms,” U.S. Pat. No. 5,786,208, the contents of whichare hereby incorporated by reference herein. This is particularlyimportant where the sample may contain components that can interferewith the amplification reaction, such as, for example, heme present in ablood sample. See Ryder et al., “Amplification of Nucleic Acids fromMononuclear Cells Using Iron Complexing and Other Agents,” U.S. Pat. No.5,639,599, the contents of which are hereby incorporated by referenceherein. Methods to prepare target nucleic acids from various sources foramplification are well known to those of ordinary skill in the art.Target nucleic acids may be purified to some degree prior to theamplification reactions described herein, but in other cases, the sampleis added to the amplification reaction without any furthermanipulations.

As will be understood by those of ordinary skill in the art, “unique”sequences are judged from the testing environment. In some embodiments,the sequences recognized by the priming oligonucleotide and/or provideroligonucleotide should be unique in the environment being tested, butneed not be unique within the universe of all possible sequences. Inother embodiments, the sequences recognized by the detection probeshould be unique in the environment being tested, but need not be uniquewithin the universe of all possible sequences. Even though the targetsequence may contain a “unique” sequence for recognition by a detectionprobe, it is not always the case that the priming oligonucleotide and/orprovider oligonucleotide are recognizing “unique” sequences. In someembodiments, it may be desirable to choose a target sequence which iscommon to a family of related organisms. In other situations, a veryhighly specific target sequence, or a target sequence having at least ahighly specific region recognized by the detection probe andamplification oligonucleotides, would be chosen so as to distinguishbetween closely related organisms.

A target sequence may be of any practical length. A minimal targetsequence includes the region which hybridizes to the primingoligonucleotide (or the complement thereof), the region which hybridizesto the hybridizing region of the provider oligonucleotide (or thecomplement thereof), and a region used for detection, e.g., a regionwhich hybridizes to a detection probe. The region which hybridizes withthe detection probe may overlap with or be contained within the regionwhich hybridizes with the priming oligonucleotide (or its complement) orthe hybridizing region of the provider oligonucleotide (or itscomplement). In addition to the minimal requirements, the optimal lengthof a target sequence depends on a number of considerations, for example,the amount of secondary structure, or self-hybridizing regions in thesequence. Typically, target sequences range from 30 nucleotides inlength to about 300 nucleotides in length. The optimal or preferredlength may vary under different conditions which can be determinedaccording to the methods described herein.

Nucleic Acid “Identity”

In certain embodiments, a nucleic acid comprises a contiguous baseregion that is at least 70%; or 75%; or 80%, or 85% or 90%, or 95%; or100% identical to a contiguous base region of a reference nucleic acid.For short nucleic acids, the degree of identity between a base region ofa “query” nucleic acid and a base region of a reference nucleic acid canbe determined by manual alignment. “Identity” is determined by comparingjust the sequence of nitrogenous bases, irrespective of the sugar andbackbone regions of the nucleic acids being compared. Thus, thequery:reference base sequence alignment may be DNA:DNA, RNA:RNA,DNA:RNA, RNA:DNA, or any combinations or analogs thereof. Equivalent RNAand DNA base sequences can be compared by converting U's (in RNA) to T's(in DNA).

Oligonucleotides & Primers

An oligonucleotide can be virtually any length, limited only by itsspecific function in the amplification reaction or in detecting anamplification product of the amplification reaction. However, in certainembodiments, preferred oligonucleotides will contain at least about 10;or 12; or 14; or 16; or 18; or 20; or 22; or 24; or 26; or 28; or 30; or32; or 34; or 36; or 38; or 40; or 42; or 44; or 46; or 48; or 50; or52; or 54; or 56 contiguous bases that are complementary to a region ofthe target nucleic acid sequence or its complementary strand. Thecontiguous bases are preferably at least about 80%, more preferably atleast about 90%, and most preferably completely complementary to thetarget sequence to which the oligonucleotide binds. Certain preferredoligonucleotides are of lengths generally between about 10-100; or12-75; or 14-50; or 15-40 bases long and optionally can include modifiednucleotides.

Oligonucleotides of a defined sequence and chemical structure may beproduced by techniques known to those of ordinary skill in the art, suchas by chemical or biochemical synthesis, and by in vitro or in vivoexpression from recombinant nucleic acid molecules, e.g., bacterial orviral vectors. As intended by this disclosure, an oligonucleotide doesnot consist solely of wild-type chromosomal DNA or the in vivotranscription products thereof.

Oligonucleotides may be modified in any way, as long as a givenmodification is compatible with the desired function of a givenoligonucleotide. One of ordinary skill in the art can easily determinewhether a given modification is suitable or desired for any givenoligonucleotide. Modifications include base modifications, sugarmodifications or backbone modifications. Base modifications include, butare not limited to the use of the following bases in addition toadenosine, cytidine, guanosine, thymidine and uridine: C-5 propyne,2-amino adenine, 5-methyl cytidine, inosine, and dP and dK bases. Thesugar groups of the nucleoside subunits may be ribose, deoxyribose andanalogs thereof, including, for example, ribonucleosides having a2′-O-methyl substitution to the ribofuranosyl moiety. See Becker et al.,U.S. Pat. No. 6,130,038. Other sugar modifications include, but are notlimited to 2′-amino, 2′-fluoro, (L)-alpha-threofuranosyl, andpentopyranosyl modifications. The nucleoside subunits may by joined bylinkages such as phosphodiester linkages, modified linkages or bynon-nucleotide moieties which do not prevent hybridization of theoligonucleotide to its complementary target nucleic acid sequence.Modified linkages include those linkages in which a standardphosphodiester linkage is replaced with a different linkage, such as aphosphorothioate linkage or a methylphosphonate linkage. The nucleobasesubunits may be joined, for example, by replacing the naturaldeoxyribose phosphate backbone of DNA with a pseudo peptide backbone,such as a 2-aminoethylglycine backbone which couples the nucleobasesubunits by means of a carboxymethyl linker to the central secondaryamine DNA analogs having a pseudo peptide backbone are commonly referredto as “peptide nucleic acids” or “PNA” and are disclosed by Nielsen etal., “Peptide Nucleic Acids,” U.S. Pat. No. 5,539,082. Other linkagemodifications include, but are not limited to, morpholino bonds.

Non-limiting examples of oligonucleotides or oligos contemplated hereininclude nucleic acid analogs containing bicyclic and tricyclicnucleoside and nucleotide analogs (LNAs). See Imanishi et al., U.S. Pat.No. 6,268,490; and Wengel et al., U.S. Pat. No. 6,670,461.) Any nucleicacid analog is contemplated by the present invention provided themodified oligonucleotide can perform its intended function, e.g.,hybridize to a target nucleic acid under stringent hybridizationconditions or amplification conditions, or interact with a DNA or RNApolymerase, thereby initiating extension or transcription. In the caseof detection probes, the modified oligonucleotides must also be capableof preferentially hybridizing to the target nucleic acid under stringenthybridization conditions.

The design and sequence of oligonucleotides depend on their function asdescribed below. Several variables to take into account include: length,melting temperature (Tm), specificity, complementarity with otheroligonucleotides in the system, G/C content, polypyrimidine (T, C) orpolypurine (A, G) stretches, and the 3′-end sequence. Controlling forthese and other variables is a standard and well known aspect ofoligonucleotide design, and various computer programs are readilyavailable to initially screen large numbers of potentialoligonucleotides.

The 3′-terminus of an oligonucleotide (or other nucleic acid) can beblocked in a variety of ways using a blocking moiety, as describedbelow. A “blocked” oligonucleotide is not efficiently extended by theaddition of nucleotides to its 3′-terminus, by a DNA- or RNA-dependentDNA polymerase, to produce a complementary strand of DNA. As such, a“blocked” oligonucleotide cannot be a “primer”.

Blocking Moiety

A blocking moiety may be a small molecule, e.g., a phosphate or ammoniumgroup, or it may be a modified nucleotide, e.g., a 3′2′dideoxynucleotide or 3′ deoxyadenosine 5′-triphosphate (cordycepin), orother modified nucleotide. Additional blocking moieties include, forexample, the use of a nucleotide or a short nucleotide sequence having a3′-to-5′ orientation, so that there is no free hydroxyl group at the3′-terminus, the use of a 3′ alkyl group, a 3′ non-nucleotide moiety(see, e.g., Arnold et al., “Non-Nucleotide Linking Reagents forNucleotide Probes,” U.S. Pat. No. 6,031,091, the contents of which arehereby incorporated by reference herein), phosphorothioate, alkane-diolresidues, peptide nucleic acid (PNA), nucleotide residues lacking a 3′hydroxyl group at the 3′-terminus, or a nucleic acid binding protein.Preferably, the 3′-blocking moiety comprises a nucleotide or anucleotide sequence having a 3′-to-5′ orientation or a 3′ non-nucleotidemoiety, and not a 3′2′-dideoxynucleotide or a 3′ terminus having a freehydroxyl group. Additional methods to prepare 3′-blockingoligonucleotides are well known to those of ordinary skill in the art.

Priming Oligonucleotide or Primer

A priming oligonucleotide is extended by the addition of covalentlybonded nucleotide bases to its 3′-terminus, which bases arecomplementary to the template. The result is a primer extension product.Suitable and preferred priming oligonucleotides are described herein.Virtually all DNA polymerases (including reverse transcriptases) thatare known require complexing of an oligonucleotide to a single-strandedtemplate (“priming”) to initiate DNA synthesis, whereas RNA replicationand transcription (copying of RNA from DNA) generally do not require aprimer. By its very nature of being extended by a DNA polymerase, apriming oligonucleotide does not comprise a 3′-blocking moiety.

Promoter Oligonucleotide/Promoter Sequence

For binding, it was generally thought that such transcriptases requiredDNA which had been rendered double-stranded in the region comprising thepromoter sequence via an extension reaction, however, it has beendetermined that efficient transcription of RNA can take place even underconditions where a double-stranded promoter is not formed through anextension reaction with the template nucleic acid. The template nucleicacid (the sequence to be transcribed) need not be double-stranded.Individual DNA-dependent RNA polymerases recognize a variety ofdifferent promoter sequences, which can vary markedly in theirefficiency in promoting transcription. When an RNA polymerase binds to apromoter sequence to initiate transcription, that promoter sequence isnot part of the sequence transcribed. Thus, the RNA transcripts producedthereby will not include that sequence.

Terminating Oligonucleotide

A terminating oligonucleotide or “blocker” is designed to hybridize tothe target nucleic acid at a position sufficient to achieve the desired3′-end for the nascent nucleic acid strand. The positioning of theterminating oligonucleotide is flexible depending upon its design. Aterminating oligonucleotide may be modified or unmodified. In certainembodiments, terminating oligonucleotides are synthesized with at leastone or more 2′-O-methyl ribonucleotides. These modified nucleotides havedemonstrated higher thermal stability of complementary duplexes. The2′-O-methyl ribonucleotides also function to increase the resistance ofoligonucleotides to exonucleases, thereby increasing the half-life ofthe modified oligonucleotides. See, e.g., Majlessi et al. (1988) NucleicAcids Res. 26, 2224-9, the contents of which are hereby incorporated byreference herein. Other modifications as described elsewhere herein maybe utilized in addition to or in place of 2′-O-methyl ribonucleotides.For example, a terminating oligonucleotide may comprise PNA or an LNA.See, e.g., Petersen et al. (2000) J. Mol. Recognit. 13, 44-53, thecontents of which are hereby incorporated by reference herein. Aterminating oligonucleotide typically includes a blocking moiety at its3′-terminus to prevent extension. A terminating oligonucleotide may alsocomprise a protein or peptide joined to the oligonucleotide so as toterminate further extension of a nascent nucleic acid chain by apolymerase. Suitable and preferred terminating oligonucleotides aredescribed herein. It is noted that while a terminating oligonucleotidetypically or necessarily includes a 3′-blocking moiety, “3′-blocked”oligonucleotides are not necessarily terminating oligonucleotides. Otheroligonucleotides as disclosed herein, e.g., provider oligonucleotidesand capping oligonucleotides are typically or necessarily 3′-blocked aswell.

Extender Oligonucleotide

An extender oligonucleotide hybridizes to a DNA template adjacent to ornear the 3′-end of the first region of a promoter oligonucleotide. Anextender oligonucleotide preferably hybridizes to a DNA template suchthat the 5′-terminal base of the extender oligonucleotide is within 3, 2or 1 bases of the 3′-terminal base of a provider oligonucleotide. Mostpreferably, the 5′-terminal base of an extender oligonucleotide isadjacent to the 3′-terminal base of a provider oligonucleotide when theextender oligonucleotide and the provider oligonucleotide are hybridizedto a DNA template. To prevent extension of an extender oligonucleotide,a 3′-terminal blocking moiety is typically included.

Probe

As would be understood by someone having ordinary skill in the art, aprobe comprises an isolated nucleic acid molecule, or an analog thereof,in a form not found in nature without human intervention (e.g.,recombined with foreign nucleic acid, isolated, or purified to someextent). Probes may have additional nucleosides or nucleobases outsideof the targeted region so long as such nucleosides or nucleobases do notsubstantially affect hybridization under stringent hybridizationconditions and, in the case of detection probes, do not preventpreferential hybridization to the target nucleic acid. Anon-complementary sequence may also be included, such as a targetcapture sequence (generally a homopolymer tract, such as a poly-A,poly-T or poly-U tail), promoter sequence, a binding site for RNAtranscription, a restriction endonuclease recognition site, or maycontain sequences which will confer a desired secondary or tertiarystructure, such as a catalytic active site or a hairpin structure on theprobe, on the target nucleic acid, or both.

The probes preferably include at least one detectable label. The labelmay be any suitable labeling substance, including but not limited to aradioisotope, an enzyme, an enzyme cofactor, an enzyme substrate, a dye,a hapten, a chemiluminescent molecule, a fluorescent molecule, aphosphorescent molecule, an electrochemiluminescent molecule, achromophore, a base sequence region that is unable to stably hybridizeto the target nucleic acid under the stated conditions, and mixtures ofthese. In one particularly preferred embodiment, the label is anacridinium ester. Certain probes as disclosed herein do not include alabel. For example, non-labeled “capture” probes may be used to enrichfor target sequences or replicates thereof, which may then be detectedby a second “detection” probe. See, e.g., Weisburg et al., “Two-StepHybridization and Capture of a Polynucleotide,” U.S. Pat. No. 6,534,273,which is hereby incorporated by reference herein. While detection probesare typically labeled, certain detection technologies do not requirethat the probe be labeled. See, e.g., Nygren et al., “Devices andMethods for Optical Detection of Nucleic Acid Hybridization,” U.S. Pat.No. 6,060,237.

Probes of a defined sequence may be produced by techniques known tothose of ordinary skill in the art, such as by chemical synthesis, andby in vitro or in vivo expression from recombinant nucleic acidmolecules. Preferably probes are 10 to 100 nucleotides in length, morepreferably 12 to 50 bases in length, and even more preferably 12 to 35bases in length.

Hybridize/Hybridization

Nucleic acid hybridization is the process by which two nucleic acidstrands having completely or partially complementary nucleotidesequences come together under predetermined reaction conditions to forma stable, double-stranded hybrid. Either nucleic acid strand may be adeoxyribonucleic acid (DNA) or a ribonucleic acid (RNA) or analogsthereof. Thus, hybridization can involve RNA:RNA hybrids, DNA:DNAhybrids, RNA:DNA hybrids, or analogs thereof. The two constituentstrands of this double-stranded structure, sometimes called a hybrid,are held together by hydrogen bonds. Although these hydrogen bonds mostcommonly form between nucleotides containing the bases adenine andthymine or uracil (A and T or U) or cytosine and guanine (C and G) onsingle nucleic acid strands, base pairing can also form between baseswhich are not members of these “canonical” pairs. Non-canonical basepairing is well-known in the art. (See, e.g., Roger L. P. Adams et al.,“The Biochemistry Of The Nucleic Acids” (11^(th) ed. 1992).

“Stringent” hybridization assay conditions refer to conditions wherein aspecific detection probe is able to hybridize with target nucleic acidsover other nucleic acids present in the test sample. It will beappreciated that these conditions may vary depending upon factorsincluding the GC content and length of the probe, the hybridizationtemperature, the composition of the hybridization reagent or solution,and the degree of hybridization specificity sought. Specific stringenthybridization conditions are provided in the disclosure below.

Nucleic Acid Amplification

Many well-known methods of nucleic acid amplification requirethermocycling to alternately denature double-stranded nucleic acids andhybridize primers; however, other well-known methods of nucleic acidamplification are isothermal. The polymerase chain reaction (U.S. Pat.Nos. 4,683,195; 4,683,202; 4,800,159; 4,965,188), commonly referred toas PCR, uses multiple cycles of denaturation, annealing of primer pairsto opposite strands, and primer extension to exponentially increase copynumbers of the target sequence. In a variation called RT-PCR, reversetranscriptase (RT) is used to make a complementary DNA (cDNA) from mRNA,and the cDNA is then amplified by PCR to produce multiple copies of DNA.The ligase chain reaction (Weiss, R. 1991, Science 254: 1292), commonlyreferred to as LCR, uses two sets of complementary DNA oligonucleotidesthat hybridize to adjacent regions of the target nucleic acid. The DNAoligonucleotides are covalently linked by a DNA ligase in repeatedcycles of thermal denaturation, hybridization and ligation to produce adetectable double-stranded ligated oligonucleotide product. Anothermethod is strand displacement amplification (Walker, G. et al., 1992,Proc. Natl. Acad. Sci. USA 89:392-396; U.S. Pat. Nos. 5,270,184 and5,455,166), commonly referred to as SDA, which uses cycles of annealingpairs of primer sequences to opposite strands of a target sequence,primer extension in the presence of a dNTPaS to produce a duplexhemiphosphorothioated primer extension product, endonuclease-mediatednicking of a hemimodified restriction endonuclease recognition site, andpolymerase-mediated primer extension from the 3′ end of the nick todisplace an existing strand and produce a strand for the next round ofprimer annealing, nicking and strand displacement, resulting ingeometric amplification of product. Thermophilic SDA (tSDA) usesthermophilic endonucleases and polymerases at higher temperatures inessentially the same method (European Pat. No. 0 684 315). Otheramplification methods include: nucleic acid sequence based amplification(U.S. Pat. No. 5,130,238), commonly referred to as NASBA; one that usesan RNA replicase to amplify the probe molecule itself (Lizardi, P. etal., 1988, BioTechnol. 6: 1197-1202), commonly referred to as Q-βreplicase; a transcription-based amplification method (Kwoh, D. et al.,1989, Proc. Natl. Acad. Sci. USA 86:1173-1177); self-sustained sequencereplication (Guatelli, J. et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878); and, transcription-mediated amplification (U.S. Pat. Nos.5,480,784 and 5,398,491), commonly referred to as TMA. For furtherdiscussion of known amplification methods see Persing, David H., 1993,“In Vitro Nucleic Acid Amplification Techniques” in Diagnostic MedicalMicrobiology: Principles and Applications (Persing et al., Eds.), pp.51-87 (American Society for Microbiology, Washington, D.C.).

In a preferred embodiment, Listeria is detected by a transcription-basedamplification technique. One preferred transcription-based amplificationsystem is transcription-mediated amplification (TMA), which employs anRNA polymerase to produce multiple RNA transcripts of a target region.Exemplary TMA amplification methods are described in U.S. Pat. Nos.5,480,784, 5,399,491, 7,374,885, and references cited therein, thecontents of which are incorporated herein by reference in theirentireties. TMA uses a “promoter-primer” that hybridizes to a targetnucleic acid in the presence of a reverse transcriptase and an RNApolymerase to form a double-stranded promoter from which the RNApolymerase produces RNA transcripts. These transcripts can becometemplates for further rounds of TMA in the presence of a second primercapable of hybridizing to the RNA transcripts. Unlike PCR, LCR or othermethods that require heat denaturation, TMA is an isothermal method thatuses an RNase H activity to digest the RNA strand of an RNA:DNA hybrid,thereby making the DNA strand available for hybridization with a primeror promoter-primer. Generally, the RNase H activity associated with thereverse transcriptase provided for amplification is used.

In one version of the TMA method, one amplification primer is anoligonucleotide promoter-primer that comprises a promoter sequence whichbecomes functional when double-stranded, located 5′ of a target-bindingsequence, which is capable of hybridizing to a binding site of a targetRNA at a location 3′ to the sequence to be amplified. A promoter-primermay be referred to as a “T7-primer” when it is specific for T7 RNApolymerase recognition. Under certain circumstances, the 3′ end of apromoter-primer, or a subpopulation of such promoter-primers, may bemodified to block or reduce promoter-primer extension. From anunmodified promoter-primer, reverse transcriptase creates a cDNA copy ofthe target RNA, while RNase H activity degrades the target RNA. A secondamplification primer then binds to the cDNA. This primer may be referredto as a “non-T7 primer” to distinguish it from a “T7-primer”. From thissecond amplification primer, reverse transcriptase creates another DNAstrand, resulting in a double-stranded DNA with a functional promoter atone end. When double-stranded, the promoter sequence is capable ofbinding an RNA polymerase to begin transcription of the target sequenceto which the promoter-primer is hybridized. An RNA polymerase uses thispromoter sequence to produce multiple RNA transcripts (i.e., amplicons),generally about 100 to 1,000 copies. Each newly-synthesized amplicon cananneal with the second amplification primer. Reverse transcriptase canthen create a DNA copy, while the RNase H activity degrades the RNA ofthis RNA:DNA duplex. The promoter-primer can then bind to the newlysynthesized DNA, allowing the reverse transcriptase to create adouble-stranded DNA, from which the RNA polymerase produces multipleamplicons. Thus, a billion-fold isothermic amplification can be achievedusing two amplification primers.

Another version of TMA uses one primer and one or more additionalamplification oligomers to amplify nucleic acids in vitro, makingtranscripts (amplicons) that indicate the presence of the targetsequence in a sample (described in Becker et al., U.S. Pat. No.7,374,885, the details of which are hereby incorporated by referenceherein). Briefly, the single-primer TMA method uses a primer (or“priming oligomer”), a modified promoter oligomer (or“promoter-provider”) that is modified to prevent the initiation of DNAsynthesis from its 3′ end (e.g., by including a 3′-blocking moiety) and,optionally, a binding molecule (e.g., a 3′-blocked extender oligomer) toterminate elongation of a cDNA from the target strand. As referred toherein, a “T7 provider” is a blocked promoter-provider oligonucleotidethat provides an oligonucleotide sequence that is recognized by T7 RNApolymerase. This method synthesizes multiple copies of a target sequenceand includes the steps of treating a target RNA that contains a targetsequence with a priming oligomer and a binding molecule, where theprimer hybridizes to the 3′ end of the target strand. RT initiatesprimer extension from the 3′ end of the primer to produce a cDNA whichis in a duplex with the target strand (e.g., RNA:cDNA). When a bindingmolecule, such as a 3′ blocked extender oligomer, is used in thereaction, it binds to the target nucleic acid adjacent near the 5′ endof the target sequence. That is, the binding molecule binds to thetarget strand next to the 5′ end of the target sequence to be amplified.When the primer is extended by DNA polymerase activity of RT to producecDNA, the 3′ end of the cDNA is determined by the position of thebinding molecule because polymerization stops when the primer extensionproduct reaches the binding molecule bound to the target strand. Thus,the 3′ end of the cDNA is complementary to the 5′ end of the targetsequence. The RNA:cDNA duplex is separated when RNase (e.g., RNase H ofRT) degrades the RNA strand, although those skilled in the art willappreciate that any form of strand separation may be used. Then, thepromoter-provider oligomer hybridizes to the cDNA near the 3′ end of thecDNA strand. The promoter-provider oligomer includes a 5′ promotersequence for an RNA polymerase and a 3′ region complementary to asequence in the 3′ region of the cDNA. The promoter-provider oligomeralso has a modified 3′ end that includes a blocking moiety that preventsinitiation of DNA synthesis from the 3′ end of the promoter-provideroligomer. In the promoter-provider:cDNA duplex, the 3′-end of the cDNAis extended by DNA polymerase activity of RT using the promoter oligomeras a template to add a promoter sequence to the cDNA and create afunctional double-stranded promoter. An RNA polymerase specific for thepromoter sequence then binds to the functional promoter and transcribesmultiple RNA transcripts complementary to the cDNA and substantiallyidentical to the target region sequence that was amplified from theinitial target strand. The resulting amplified RNA can then cyclethrough the process again by binding the primer and serving as atemplate for further cDNA production, ultimately producing manyamplicons from the initial target nucleic acid present in the sample.Some embodiments of the single-primer transcription-associatedamplification method do not include the binding molecule and, therefore,the cDNA product made from the primer has an indeterminate 3′ end, butthe amplification steps proceed substantially as described above for allother steps.

Suitable amplification conditions can be readily determined by a skilledartisan in view of the present disclosure. “Amplification conditions” asdisclosed herein refer to conditions which permit nucleic acidamplification. Amplification conditions may, in some embodiments, beless stringent than “stringent hybridization conditions” as describedherein. Oligonucleotides used in the amplification reactions asdisclosed herein may be specific for and hybridize to their intendedtargets under amplification conditions, but in certain embodiments mayor may not hybridize under more stringent hybridization conditions. Onthe other hand, detection probes generally hybridize under stringenthybridization conditions. While the Examples section infra providespreferred amplification conditions for amplifying target nucleic acidsequences, other acceptable conditions to carry out nucleic acidamplifications could be easily ascertained by someone having ordinaryskill in the art depending on the particular method of amplificationemployed.

The amplification methods as disclosed herein, in certain embodiments,also preferably employ the use of one or more other types ofoligonucleotides that are effective for improving the sensitivity,selectivity, efficiency, etc., of the amplification reaction. These mayinclude, for example, terminating oligonucleotides, extender, and/orhelper oligonucleotides, and the like.

Target Capture

In certain embodiments, it may be preferred to purify or enrich a targetnucleic acid from a sample prior to amplification, for example using atarget capture approach. “Target capture” (TC) refers generally tocapturing a target polynucleotide onto a solid support, such asmagnetically attractable particles, wherein the solid support retainsthe target polynucleotide during one or more washing steps of the targetpolynucleotide purification procedure. In this way, the targetpolynucleotide is substantially purified prior to a subsequent nucleicacid amplification step. Numerous target capture methods are known andsuitable for use in conjunction with the methods described herein.

Any support may be used, e.g., matrices or particles free in solution,which may be made of any of a variety of materials, e.g., nylon,nitrocellulose, glass, polyacrylate, mixed polymers, polystyrene, silanepolypropylene, or metal. Illustrative examples use a support that ismagnetically attractable particles, e.g., monodisperse paramagneticbeads (uniform size.+−.5%) to which an immobilized probe is joineddirectly (e.g., via covalent linkage, chelation, or ionic interaction)or indirectly (e.g., via a linker), where the joining is stable duringnucleic acid hybridization conditions.

For example, one illustrative approach, as described in U.S. PatentApplication Publication No 20060068417, uses at least one capture probeoligonucleotide that contains a target-complementary region and a memberof a specific binding pair that attaches the target nucleic acid to animmobilized probe on a capture support, thus forming a capture hybridthat is separated from other sample components before the target nucleicacid is released from the capture support.

In another illustrative method, Weisburg et al., in U.S. Pat. No.6,110,678, describe a method for capturing a target polynucleotide in asample onto a solid support, such as magnetically attractable particles,with an attached immobilized probe by using a capture probe and twodifferent hybridization conditions, which preferably differ intemperature only. The two hybridization conditions control the order ofhybridization, where the first hybridization conditions allowhybridization of the capture probe to the target polynucleotide, and thesecond hybridization conditions allow hybridization of the capture probeto the immobilized probe. The method may be used to detect the presenceof a target polynucleotide in a sample by detecting the captured targetpolynucleotide or amplified target polynucleotide.

Another illustrative target capture technique (U.S. Pat. No. 4,486,539)involves a hybridization sandwich technique for capturing and fordetecting the presence of a target polynucleotide. The techniqueinvolves the capture of the target polynucleotide by a probe bound to asolid support and hybridization of a detection probe to the capturedtarget polynucleotide. Detection probes not hybridized to the targetpolynucleotide are readily washed away from the solid support. Thus,remaining label is associated with the target polynucleotide initiallypresent in the sample.

Another illustrative target capture technique (U.S. Pat. No. 4,751,177)involves a method that uses a mediator polynucleotide that hybridizes toboth a target polynucleotide and to a polynucleotide fixed on a solidsupport. The mediator polynucleotide joins the target polynucleotide tothe solid support to produce a bound target. A labeled probe can behybridized to the bound target and unbound labeled probe can be washedaway from the solid support.

Yet another illustrative target capture technique is described in U.S.Pat. Nos. 4,894,324 and 5,288,609, which describe a method for detectinga target polynucleotide. The method utilizes two single-strandedpolynucleotide segments complementary to the same or opposite strands ofthe target and results in the formation of a double hybrid with thetarget polynucleotide. In one embodiment, the hybrid is captured onto asupport.

In another illustrative target capture technique, EP Pat. Pub. No. 0 370694, methods and kits for detecting nucleic acids use oligonucleotideprimers labeled with specific binding partners to immobilize primers andprimer extension products. The label specifically complexes with itsreceptor which is bound to a solid support.

The above capture techniques are illustrative only, and not limiting.Indeed, essentially any technique available to the skilled artisan maybe used provided it is effective for purifying a target nucleic acidsequence of interest prior to amplification.

Nucleic Acid Detection

Essentially any labeling and/or detection system that can be used formonitoring specific nucleic acid hybridization can be used inconjunction to detect Listeria amplicons. Many such systems are knownand available to the skilled artisan, illustrative examples of which arebriefly discussed below.

Detection systems typically employ a detection oligonucleotide of onetype or another in order to facilitate detection of the target nucleicacid of interest. Detection may either be direct (i.e., probe hybridizeddirectly to the target) or indirect (i.e., a probe hybridized to anintermediate structure that links the probe to the target). A probe'starget sequence generally refers to the specific sequence within alarger sequence which the probe hybridizes specifically. A detectionprobe may include target-specific sequences and other sequences orstructures that contribute to the probe's three-dimensional structure,depending on whether the target sequence is present (e.g., U.S. Pat.Nos. 5,118,801, 5,312,728, 6,835,542, and 6,849,412).

Any of a number of well known labeling systems may be used to facilitatedetection. Direct joining may use covalent bonds or non-covalentinteractions (e.g., hydrogen bonding, hydrophobic or ionic interactions,and chelate or coordination complex formation) whereas indirect joiningmay use a bridging moiety or linker (e.g., via an antibody or additionaloligonucleotide(s), which amplify a detectable signal. Any detectablemoiety may be used, e.g., radionuclide, ligand such as biotin or avidin,enzyme, enzyme substrate, reactive group, chromophore such as a dye orparticle (e.g., latex or metal bead) that imparts a detectable color,luminescent compound (e.g. bioluminescent, phosphorescent orchemiluminescent compound), and fluorescent compound. Preferredembodiments include a “homogeneous detectable label” that is detectablein a homogeneous system in which bound labeled probe in a mixtureexhibits a detectable change compared to unbound labeled probe, whichallows the label to be detected without physically removing hybridizedfrom unhybridized labeled probe (e.g., U.S. Pat. Nos. 6,004,745,5,656,207 and 5,658,737). Preferred homogeneous detectable labelsinclude chemiluminescent compounds, more preferably acridinium ester(“AE”) compounds, such as standard AE or AE derivatives which are wellknown (U.S. Pat. Nos. 5,656,207, 5,658,737, and 5,948,899). Methods ofsynthesizing labels, attaching labels to nucleic acid, and detectingsignals from labels are well known (e.g., Sambrook et al., MolecularCloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989) at Chapter. 10, and U.S. Pat.Nos. 6,414,152, 5,185,439, 5,658,737, 5,656,207, 5,547,842, 5,639,604,4,581,333, and 5,731,148). Preferred methods of linking an AE compoundto a nucleic acid are known (e.g., U.S. Pat. No. 5,585,481 and U.S. Pat.No. 5,639,604, see column 10, line 6 to column 11, line 3, and Example8). Preferred AE labeling positions are a probe's central region andnear a region of A/T base pairs, at a probe's 3′ or 5′ terminus, or ator near a mismatch site with a known sequence that the probe should notdetect compared to the desired target sequence.

In a preferred embodiment, oligonucleotides exhibiting at least somedegree of self-complementarity are desirable to facilitate detection ofprobe:target duplexes in a test sample without first requiring theremoval of unhybridized probe prior to detection. By way of example,when exposed to denaturing conditions, the two complementary regions ofa molecular torch, which may be fully or partially complementary, melt,leaving the target binding domain available for hybridization to atarget sequence when the predetermined hybridization assay conditionsare restored. Molecular torches are designed so that the target bindingdomain favors hybridization to the target sequence over the targetclosing domain. The target binding domain and the target closing domainof a molecular torch include interacting labels (e.g., afluorescent/quencher pair) positioned so that a different signal isproduced when the molecular torch is self-hybridized as opposed to whenthe molecular torch is hybridized to a target nucleic acid, therebypermitting detection of probe:target duplexes in a test sample in thepresence of unhybridized probe having a viable label associatedtherewith. Molecular torches are fully described in U.S. Pat. No.6,361,945, the disclosure of which is hereby incorporated by referenceherein.

Another example of a self-complementary hybridization assay probe thatmay be used is a structure commonly referred to as a “molecular beacon.”Molecular beacons comprise nucleic acid molecules having a targetcomplementary sequence, an affinity pair (or nucleic acid arms) thatholds the probe in a closed conformation in the absence of a targetnucleic acid sequence, and a label pair that interacts when the probe isin a closed conformation. Hybridization of the molecular beacon targetcomplementary sequence to the target nucleic acid separates the membersof the affinity pair, thereby shifting the probe to an openconformation. The shift to the open conformation is detectable due toreduced interaction of the label pair, which may be, for example, afluorophore and a quencher (e.g., DABCYL and EDANS). Molecular beaconsare fully described in U.S. Pat. No. 5,925,517, the disclosure of whichis hereby incorporated by reference herein. Molecular beacons useful fordetecting specific nucleic acid sequences may be created by appending toeither end of one of the probe sequences disclosed herein, a firstnucleic acid arm comprising a fluorophore and a second nucleic acid armcomprising a quencher moiety. In this configuration, Listeria-specificprobe sequences may serve as the target-complementary “loop” portion ofthe resulting molecular beacon.

Molecular beacons are preferably labeled with an interactive pair ofdetectable labels. Preferred detectable labels interact with each otherby FRET or non-FRET energy transfer mechanisms. Fluorescence resonanceenergy transfer (FRET) involves the radiationless transmission of energyquanta from the site of absorption to the site of its utilization in themolecule or system of molecules by resonance interaction betweenchromophores, over distances considerably greater than interatomicdistances, without conversion to thermal energy, and without the donorand acceptor coming into kinetic collision. The “donor” is the moietythat initially absorbs the energy, and the “acceptor” is the moiety towhich the energy is subsequently transferred. In addition to FRET, thereare at least three other “non-FRET” energy transfer processes by whichexcitation energy can be transferred from a donor to an acceptormolecule.

When two labels are held sufficiently close such that energy emitted byone label can be received or absorbed by the second label, whether by aFRET or non-FRET mechanism, the two labels are said to be in an “energytransfer relationship.” This is the case, for example, when a molecularbeacon is maintained in the closed state by formation of a stem duplexand fluorescent emission from a fluorophore attached to one arm of themolecular beacon is quenched by a quencher moiety on the other arm.

Illustrative label moieties for the molecular beacons include afluorophore and a second moiety having fluorescence quenching properties(i.e., a “quencher”). In this embodiment, the characteristic signal islikely fluorescence of a particular wavelength, but alternatively couldbe a visible light signal. When fluorescence is involved, changes inemission are preferably due to FRET, or to radiative energy transfer ornon-FRET modes. When a molecular beacon having a pair of interactivelabels in the closed state is stimulated by an appropriate frequency oflight, a fluorescent signal is generated at a first level, which may bevery low. When this same molecular beacon is in the open state and isstimulated by an appropriate frequency of light, the fluorophore and thequencher moieties are sufficiently separated from each other such thatenergy transfer between them is substantially precluded. Under thatcondition, the quencher moiety is unable to quench the fluorescence fromthe fluorophore moiety. If the fluorophore is stimulated by light energyof an appropriate wavelength, a fluorescent signal of a second level,higher than the first level, will be generated. The difference betweenthe two levels of fluorescence is detectable and measurable. Usingfluorophore and quencher moieties in this manner, the molecular beaconis only “on” in the “open” conformation and indicates that the probe isbound to the target by emanating an easily detectable signal. Theconformational state of the probe alters the signal generated from theprobe by regulating the interaction between the label moieties.

Examples of donor/acceptor label pairs that may be used, making noattempt to distinguish FRET from non-FRET pairs, includefluorescein/tetramethylrhodamine, IAEDANS/fluorescein, EDANS/DABCYL,coumarin/DABCYL, fluorescein/fluorescein, BODIPY FL/BODIPY FL,fluorescein/DABCYL, lucifer yellow/DABCYL, BODIPY/DABCYL, eosine/DABCYL,erythrosine/DABCYL, tetramethylrhodamine/DABCYL, Texas Red/DABCYL,CY5/BH1, CY5/BH2, CY3/BH1, CY3/BH2, and fluorescein/QSY7 dye. Thosehaving an ordinary level of skill in the art will understand that whendonor and acceptor dyes are different, energy transfer can be detectedby the appearance of sensitized fluorescence of the acceptor or byquenching of donor fluorescence. When the donor and acceptor species arethe same, energy can be detected by the resulting fluorescencedepolarization. Non-fluorescent acceptors such as DABCYL and the QSY 7dyes advantageously eliminate the potential problem of backgroundfluorescence resulting from direct (i.e., non-sensitized) acceptorexcitation. Preferred fluorophore moieties that can be used as onemember of a donor-acceptor pair include fluorescein, ROX, and the CYdyes (such as CY5). Highly preferred quencher moieties that can be usedas another member of a donor-acceptor pair include DABCYL and the BlackHole Quencher moieties, which are available from Biosearch Technologies,Inc. (Novato, Calif.).

Synthetic techniques and methods of attaching labels to nucleic acidsand detecting labels are well known in the art (see, e.g., Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989), Chapter 10; Nelson etal., U.S. Pat. No. 5,658,737; Woodhead et al., U.S. Pat. No. 5,656,207;Hogan et al., U.S. Pat. No. 5,547,842; Arnold et al., U.S. Pat. Nos.5,185,439 and 6,004,745; Kourilsky et al., U.S. Pat. No. 4,581,333; and,Becker et al., U.S. Pat. No. 5,731,148).

Preferred Listeria Oligonucleotides and Oligonucleotide Sets

As described herein, preferred sites for amplifying and detectingListeria nucleic acids as disclosed herein have been found to reside inthe 450 region of Listeria 16S rRNA. Moreover, particularly preferredoligonucleotides and oligonucleotide sets within this region have beenidentified for amplifying Listeria 16S with improved sensitivity,selectivity and specificity. It will be understood that theoligonucleotides disclosed herein are capable of hybridizing to aListeria target sequence with high specificity and, as a result, arecapable of participating in a nucleic acid amplification reaction thatcan be used to detect the presence and/or levels of one or more Listeriaspecies in a sample and distinguish it from the presence of otherclosely related species.

For example, in one embodiment, the amplification oligonucleotidescomprise a first oligonucleotide and a second oligonucleotide, whereinthe first and second oligonucleotides target the 450 region of theListeria 16S rRNA with a high degree of specificity.

The amplification oligonucleotides disclosed herein are particularlyeffective for amplifying a target nucleic acid sequence of Listeria in atranscription-based amplification reaction, preferably a real-timetranscription-mediated amplification (TMA) reaction.

It will be understood that in addition to the particular T7 provideroligonucleotides and primer oligonucleotides used in the amplificationreaction, additional oligonucleotides will also generally be employed inconjunction with the amplification reaction. For example, in certainembodiments, the amplification reactions will also employ the use of oneor more of a detection oligonucleotide (e.g., a torch oligonucleotide oran acridinium ester probe), and a blocker oligonucleotide.

Table 1 presents specific examples of T7 Provider oligonucleotides,Primer oligonucleotides, and other ancillary oligonucleotides (e.g.,Blocker, Torch, Target Capture, AE probes, and Helper oligonucleotides)that have been identified for the 450 region by the invention.

TABLE 1 Examples of Preferred Oligonucleotides16S rRNA 450 Region Sequences Use SEQ ID NO: 450 Region Sequence BlockerSEQ ID NO: 1 cauugcggaagauucccuac-X Blocker SEQ ID NO: 2cguccauugcggaagauucc-X Blocker SEQ ID NO: 3 cuuucguccauugcggaagauuc-XBlocker SEQ ID NO: 4 cagacuuucguccauugcggaag-X Blocker SEQ ID NO: 5guugcuccgucagacuuucgucc-X Blocker SEQ ID NO: 6 gcggcguugcuccgucagac-XBlocker SEQ ID NO: 7 ccuucuucauacacgcgg-X T7 SEQ ID NO: 8AATTTAATACGACTCACTATAGGGAGACAATGGAC Provider GAAAGTCTGACGGAGC-X T7SEQ ID NO: 9 AATTTAATACGACTCACTATAGGGAGAGGACGAAA ProviderGTCTGACGGAGCAACG-X T7 SEQ ID NO: 10 AATTTAATACGACTCACTATAGGGAGAGAAAGTCTProvider GACGGAGCAACGCCGC-X T7 SEQ ID NO: 11AATTTAATACGACTCACTATAGGGAGAGTCTGACG Provider GAGCAACGCCGCGTG-X T7SEQ ID NO: 12 AATTTAATACGACTCACTATAGGGAGAGCAACGCC ProviderGCGTGTATGAAGAAGG-X T7 SEQ ID NO: 13 AATTTAATACGACTCACTATAGGGAGAGCCGCGTGProvider TATGAAGAAGG-X T7 SEQ ID NO: 14AATTTAATACGACTCACTATAGGGAGAGCCGCGTG Provider TGTGAAGAAGG-X T7SEQ ID NO: 15 AATTTAATACGACTCACTATAGGGAGAGAAGGTTT ProviderTCGGATCGTAAAG-X Primer SEQ ID NO: 16 CAAGCAGTTACTCTTATCCTTGTTCTTCTCPrimer SEQ ID NO: 17 GGGACAAGCAGTTACTCTTATCC Primer SEQ ID NO: 18CCGTCAAGGGACAAGCAGTTACTC Primer SEQ ID NO: 19 GATACCGTCAAGGGACAAGCPrimer SEQ ID NO: 20 GGTTAGATACCGTCAAGGGACAAGC Primer SEQ ID NO: 21TTAGATACCGTCAAGGGACA Primer SEQ ID NO: 22 GGTTAGATACCGTCAAGGGACA PrimerSEQ ID NO: 23 GGCTTTCTGGTTAGATACCGTC Torch SEQ ID NO: 24cccaguacuuuacgauccgcuggg Torch SEQ ID NO: 25 ccggcaguacuuuacgauccggTorch SEQ ID NO: 26 ccggacaguacuuuacgauccgg Torch SEQ ID NO: 27ggcaguuacucuuauccuugcugcc Torch SEQ ID NO: 28 gggacaagcaguuacgucccTarget SEQ ID NO: 29 ccaacuagcuaaugcaccgcgggcTTTAAAAAAAAAAAAAA CaptureAAAAAAAAAAAAAAAA Target SEQ ID NO: 30ccattaccctaccaactagctaatgcaccgTTTAAAAAAAAAAAA Capture AAAAAAAAAAAAAAAAAATarget SEQ ID NO: 31 ccauuacccuaccaacuagcuaaugcTTTAAAAAAAAAAAA CaptureAAAAAAAAAAAAAAAAAA Target SEQ ID NO: 32gggccgugucucagucccaguguggTTTAAAAAAAAAAAAA Capture AAAAAAAAAAAAAAAAATarget SEQ ID NO: 33 cugccucccguaggagucugggcTTTAAAAAAAAAAAAAAA CaptureAAAAAAAAAAAAAAA Target SEQ ID NO: 34gcacguaguuagccguggcuuucuggTTTAAAAAAAAAAAA Capture AAAAAAAAAAAAAAAAAATarget SEQ ID NO: 35 gctgctggcacgtagttagccgtgTTTAAAAAAAAAAAAAAAA CaptureAAAAAAAAAAAAAA Target SEQ ID NO: 36gcugcuggcacguaguuagccgugTTTAAAAAAAAAAAAAA Capture AAAAAAAAAAAAAAAA “X” =optional blocking moiety (e.g., reverse 3′-5′-C); lower case n =2′-O-methyl ribose; upper case N = deoxyribose; 5′-fluorescein (“F”)fluorophore and 3′-dabsyl (“D”) quencher moieties were attached to thetorch oligonucleotides

In addition, Table 2 identifies two particularly preferredoligonucleotide sets for use in the compositions, kits and methods asdisclosed herein.

TABLE 2 Example of Two Preferred Oligonucleotide Sets 450 RegionOligonucleotide Set Description Oligonucleotide Set #1 T7 Provider SEQID NO: 13 Blocker SEQ ID NO: 6 Primer SEQ ID NO: 23 Torch SEQ ID NO: 27Set #2 T7 Provider SEQ ID NO: 14 Blocker SEQ ID NO: 6 Primer SEQ ID NO:23 Torch SEQ ID NO: 27

While specifically preferred amplification oligonucleotides derived fromthe 450 region have been identified, which result in superior assayperformance, it will be recognized that other oligonucleotides derivedfrom the 450 region and having insubstantial modifications from thosespecifically described herein may also be used, provided the same orsimilar performance objectives are achieved. For example,oligonucleotides derived from the 450 region and useful in theamplification reactions as disclosed herein can have different lengthsfrom those identified herein, provided it does not substantially affectamplification and/or detection procedures. These and other routine andinsubstantial modifications to the preferred oligonucleotides can becarried out using conventional techniques, and to the extent suchmodifications maintain one or more advantages provided herein they areconsidered within the spirit and scope of the invention.

The general principles as disclosed herein may be more fully appreciatedby reference to the following non-limiting Examples.

EXAMPLES

Examples are provided below illustrating certain aspects andembodiments. The examples below are believed to accurately reflect thedetails of experiments actually performed, however, it is possible thatsome minor discrepancies may exist between the work actually performedand the experimental details set forth below which do not affect theconclusions of these experiments or the ability of skilled artisans topractice them. Skilled artisans will appreciate that these examples arenot intended to limit the invention to the specific embodimentsdescribed therein. Additionally, those skilled in the art, using thetechniques, materials and methods described herein, could easily deviseand optimize alternative amplification systems for carrying out theseand related methods while still being within the spirit and scope of thepresent invention.

Unless otherwise indicated, oligonucleotides and modifiedoligonucleotides in the following examples were synthesized usingstandard phosphoramidite chemistry, various methods of which are wellknown in the art. See e.g., Carruthers, et al., 154 Methods inEnzymology, 287 (1987), the contents of which are hereby incorporated byreference herein. Unless otherwise stated herein, modified nucleotideswere 2′-O-methyl ribonucleotides, which were used in the synthesis astheir phosphoramidite analogs. For blocked oligonucleotides used insingle-primer amplification (Becker et al., U.S. Pat. No. 7,374,885,hereby incorporated by reference herein), the 3′-terminal blockingmoiety consisted of a “reversed C” 3′-to-3′ linkage prepared using3′-dimethyltrityl-N-benzoyl-2′-deoxycytidine, 5′-succinoyl-long chainalkylamino-CPG (Glen Research Corporation, Cat. No. 20-0102-01).Molecular torches (see Becker et al., U.S. Pat. No. 6,849,412, herebyincorporated by reference herein) were prepared using a C9non-nucleotide (triethylene glycol) linker joining region (SpacerPhosphoramidite 9, Glen Research Corporation, Cat. No. 10-1909-xx),5′-fluorescein (“F”) fluorophore and 3′-dabsyl (“D”) quencher moietiesattached to the oligonucleotide by standard methods.

As set forth in the examples below, analyses of a wide variety ofamplification reagents and conditions has led to the development of ahighly sensitive and selective amplification process for the detectionof Listeria.

Example 1 Description of Illustrative Assay Reagents, Equipment andMaterials

The following example describes typical assay reagents, protocols,conditions and the like used in the real-time TMA experiments describedherein. Unless specified to the contrary, reagent preparation, equipmentpreparation and assay protocols were performed essentially as set forthbelow.

L. monocytogenes, ATCC 35152, was used as the positive control in allruns. Amplification Reagent was made ahead of time.

A. Reagents and Samples

1. Amplification Reagent. The “Amplification Reagent” or “Amp Reagent”comprised approximate concentrations of the following components: 0.5 mMdATP, 0.5 mM dCTP, 0.5 mM dGTP, 0.5 mM dTTP, 10 mM ATP, 2 mM CTP, 2 mMGTP, 12.7 mM UTP, 30 mM MgCl₂, and 33 mM KCl in 50 mM HEPES buffer at pH7.7. Primers and other oligonucleotides were added to the Amp Reagent.

2. Enzyme Reagent. The “Enzyme Reagent” comprised approximateconcentrations of the following components: 1180 RTU/μL Moloney murineleukemia virus (“MMLV”) reverse transcriptase (“RT”) and 260 PU/μL T7RNA polymerase in 75 mM HEPES buffer containing 120 mM KCl, 10% TRITON®X-100, 160 mM N-acetyl-L-cysteine, and 1 mM EDTA at pH 7.0, where oneRTU of RT activity incorporates 1 nmol of dT into a substrate in 20minutes at 37° C. and one PU of T7 RNA polymerase activity produces 5fmol of RNA transcript in 20 minutes at 37° C.

3. Wash Solution. The “Wash Solution” comprised 0.1% (w/v) sodiumdodecyl sulfate, 150 mM NaCl and 1 mM EDTA in 10 mM HEPES buffer at pHto 7.5.

4. Target Capture Reagent. The “Target Capture Reagent” (TCR) comprisedapproximate concentrations of the following components: 60 pmol/mL eachof one or more capture probes having a dT₃dA₃₀ tail and an optionalcapture helper probe, 250 to 300 ug/mL paramagnetic oligo-(dT)₁₄microparticles (Seradyn), 250 mM HEPES, 100 mM EDTA and 1.88 M LiCl atpH 6.5.

5. Lysis Reagent. The “Lysis Buffer” comprised 1% lithium lauryl sulfatein a buffer containing 100 mM tris, 2.5 mM succinic acid, 10 mM EDTA and500 mM LiCl at pH 6.5.

6. Target rRNA Samples. rRNA samples were stored in water, 0.1% LiLS orLysis Reagent prior to use in the experiments described herein.

B. Equipment and Materials

-   -   KingFisher 96 Processor (“KF96”) (Thermo Fisher Scientific,        Waltham, Mass.).    -   KingFisher® 96 tip comb for DW magnets (Thermo Fisher Scientific        catalog no. 97002534).    -   KingFisher® 96 KF plate (200 microliters) (Thermo Fisher        Scientific catalog no. 97002540).    -   Hard-Shell Thin-Wall 96-Well Skirted PCR Plate, colored        shell/white well (“MJ plate”) (catalog numbers: HSP-9615,        HSP-9625, HSP-9635, Bio-Rad Laboratories, Hercules, Calif.).    -   DW 96 plate, V bottom, Polypropylene, sterile 25 pcs/case        (Axygen catalog no. P-2ML-SQ-C-S; VWR catalog no. 47749-874;        Thermo Fisher Scientific catalog no. 95040460).    -   eppendorf Thermomixer® R Dry Block Heating and Cooling Shaker        with cat. no. 022670565 thermoblock (Eppendorf Corporation,        Westbury, N.Y.).    -   FLUOstar fluorescence microplate reader (BMG Labtech Inc., Cary,        N.C.).    -   PTI® FluoDia® T70 micoplate fluorometer. (Photon Technology        International Inc., Birmingham, N.J.)

Example 2 Design and Evaluation of Listeria Oligonucleotide Sets

Using a region corresponding to the 450 region of the E. coli rRNAsequence (FIG. 2), several T7 Providers, Blockers, Primers, and Torcheswere designed.

A. Screening

The real-time TMA amplification reactions were performed essentially asfollows for simultaneous amplification and detection of analyte and theinternal control. A “masterplate” was made that contained T7 Provider(0.5 pmol/μL), Primer (0.5 pmol/μL), and Blocker (0.05 pmol/μL) inreconstituted oligoless amplification reagent (Amp Reagent). Eachmasterplate well contained 200 μL of a different combination of oligos.For testing, 10 μL of each masterplate well mixture was placed in an MJplate for amplification. 20 μL of the target nucleic acid in oligolessamplification reagent was added to the plate. Amplification reagentwithout target was used as the negative control. The amplificationplates were covered and placed on a THERMOMIXER apparatus at 60° C. for10 minutes to anneal the Primer. The plates were cooled to 42° C. on theTHERMOMIXER apparatus and incubated for an additional 15 minutes. Theplates were then uncovered and 10 μL of enzyme reagent containingdetection probe at a concentration of about 1 pmol/μL was added to eachamplification reaction well. The plates were covered and mixed on theTHERMOMIXER apparatus at 1400 rpm for 1 minute. The plates wereimmediately placed in a BMG microplate fluorometer and the assay run.Reads were taken every 72 seconds for a total of 63 reads. Two colorswere read for each interval. Examples of real-time TMA assay curves areshown in FIG. 1. Assay curve plots and TTime analysis were used toanalyze the data.

B. Best Combinations

The best combinations from the screening with L. monocytogenes are shownin Table 3. They were chosen on the basis of fluorescence emergence timeand maximum fluorescent signal. The oligonucleotide sets were thentested for sensitivity with L. innocua, L. grayi, L. ivanovii, L.welshimeri, L. murrayi, and L. seeligeri. The oligonucleotide sets werealso tested for specificity against Brochothrix thermosphacta andErysipelothrix rhusiopathiae. Each target was tested at a target levelof 0 or 1E5 copies/assay.

TABLE 3 Listeria Oligonucleotide Sets 450 Region Oligonucleotide SetDescription Oligonucleotide Set #1 T7 Provider SEQ ID NO: 15 Blocker SEQID NO: 7 Primer SEQ ID NO: 23 Torch SEQ ID NO: 27 Set #2 T7 Provider SEQID NO: 12 Blocker SEQ ID NO: 5 Primer SEQ ID NO: 23 Torch SEQ ID NO: 27Set #3 T7 Provider SEQ ID NO: 13 Blocker SEQ ID NO: 6 Primer SEQ ID NO:23 Torch SEQ ID NO: 27 Set #4 T7 Provider SEQ ID NO: 15 Blocker SEQ IDNO: 7 Primer SEQ ID NO: 21 Torch SEQ ID NO: 27 Set #5 T7 Provider SEQ IDNO: 12 Blocker SEQ ID NO: 5 Primer SEQ ID NO: 21 Torch SEQ ID NO: 27 Set#6 T7 Provider SEQ ID NO: 13 Blocker SEQ ID NO: 6 Primer SEQ ID NO: 21Torch SEQ ID NO: 27 Set #7 T7 Provider SEQ ID NO: 12 Blocker SEQ ID NO:5 Primer SEQ ID NO: 23 Torch SEQ ID NO: 25 Set #8 T7 Provider SEQ ID NO:13 Blocker SEQ ID NO: 6 Primer SEQ ID NO: 23 Torch SEQ ID NO: 25 Set #9T7 Provider SEQ ID NO: 8 Blocker SEQ ID NO: 1 Primer SEQ ID NO: 23 TorchSEQ ID NO: 25 Set #10 T7 Provider SEQ ID NO: 12 Blocker SEQ ID NO: 5Primer SEQ ID NO: 16 Torch SEQ ID NO: 25 Set #11 T7 Provider SEQ ID NO:13 Blocker SEQ ID NO: 6 Primer SEQ ID NO: 16 Torch SEQ ID NO: 25 Set #12T7 Provider SEQ ID NO: 8 Blocker SEQ ID NO: 1 Primer SEQ ID NO: 16 TorchSEQ ID NO: 25

Set #3 was chosen for further evaluation. Set #6 was chosen as theback-up as it only differed in the choice of primer component. Theback-up set had no cross-reaction with either of the nearest neighborsbut did not perform as well on the L. grayi and L. murrayi species.Other sets were excluded based on either poor performance to all theListeria sp. Or cross-reaction observed in either E. rhusiopathiae or B.thermosphacta.

Example 3 Evaluation of Target Capture Integration

Target capture oligos (TCO) for the Listeria 16S rRNA were designed tocapture target rRNA in regions corresponding from about 230 to 355 andfrom about 490 to about 525 bp of E. coli rRNA. Eight oligos (seeTable 1) were designed, synthesized, and tested. Specificity was notbuilt into the TCO's, therefore, they were screened using only L.monocytogenes and L. grayi RNA. The KINGFISHER 96 processor (“KF96”) wasused with a large magnet. Target capture was performed using an Axygendeep-well (DW 96) plate using a 1 mL sample volume. Analysis of the L.monocytogenes capture showed that TCOs 2, 3, 6, and 7 were slightlybetter than TCO 1, 4, 5, and 8. Further evaluation demonstrated thatTCOs 2, 3, 6, and 7 all worked acceptably well. Therefore, it wasdecided to scale-up TCO 2 for the Food Study.

The method of Target Capture with the KINGFISHER 96 processor issummarized in Table 4. In brief, samples were mixed with Lysis Reagentto release target and stabilize rRNA. Target Capture Reagent was added.Ribosomal RNA target was captured and purified on magnetic particlesusing the KINGFISHER 96 purification system. Particles were resuspendedin Amplification Reagent containing 6-carboxyfluoroscein (“FAM”)-labeledTorch for analyte and 6-carboxy-tetramethyl-rhodamine (“TAMRA”)-labeledTorch for the internal control. A typical target capture procedure topurify and prepare nucleic acid samples for subsequent amplification wasperformed essentially as described below. 100 μL of test sample, 50 μLof the TCR containing target capture oligonucleotides, and 1 mL LysisReagent were combined and incubated at 60° C. for 15 minutes. The TCRmagnetic particles from the treated reaction mixture were captured andwashed using the Wash Solution and a suitable magnetic particle washingand separation device (e.g., a magnetic separation rack, a GEN-PROBETarget Capture System (Gen-Probe Cat. No. 5207) or a KINGFISHER magneticparticle processor system (available from Thermo Fisher Scientific).After washing, the magnetic particles were resuspended in 100 μL of theAmplification Reagent.

Specifically, frozen amplification reagent was thawed and lyophilizedenzyme reagent was reconstituted. A wash plate was prepared by filling aKF200 plate with 200 μL/well of wash solution. An amp plate was preparedby filling another KF200 plate with 100 μL/well of amplificationreagent. Both the amp and wash plates were covered until used. A sampleplate was prepared by adding 50 μL non-specific TCR/well into a 2-mL,deep-well 96 plate (Axygen). The target was diluted to the requiredconcentrations in 10 μL lysis solution. One ml of lysis solution wasadded to each well of the sample plate. With a repeat pipettor, 10 μL oftarget solution was added to the appropriate deep wells. A deep-welltip-comb was placed in the sample plate. The covers for the wash and ampplates were removed. The KF96 protocol was started and all three plateswere placed on the KF96 instrument. The amp plate was placed in position3, the wash plate in position 2, and the sample (deep-well plate) inposition 1. Once the plates were loaded, the KF96 instrument began thetarget capture step. When the KF96 run was completed, the plates wereremoved. From the amp plate, 30 μL from each well were removed using amulti-channel pipettor and transferred to an MJ 96-well PCR plate.

TABLE 4 KINGFISHER 96 Program Step Step Position Description ActionBeginning Mix End 1 1 Capture Mix No action Very Collect slow-30 beadsminutes 20 times 2 2 Release to Wash Release 30 s Slow No Wash 30 s Slowaction 3 1 2^(nd) Sample Collect Collect No No Collection beads actionAction 20 times 4 2 Release to Wash Release 30 s very Collect Wash 2 30s Slow Slow beads- count 20 5 3 Release into Elution Release 30 s SlowNo Amp Soln 30 s Slow action

Example 4 Sensitivity, Specificity, and Interference EvaluationSensitivity

L. monocytogenes (ATCC 35152), L. grayi (ATCC 19120), L. innocua (ATCC33090), L. ivanovii (F4081), L. murrayi (F4076), L. seeligeri (F4088),and L. welshimeri (F4082) were assayed at 1E5 copies/reaction. Lysisbuffer was used as the negative control. Twenty reactions of each weretested using the KINGFISHER 96 instrument for target capture and the BMGreader for detection. From each reaction, one 30 uL replicate wasamplified. The positive criterion was 3000 RFU. Nineteen of 20replicates were to be detected with >95% positivity rate. The results oftesting for Sensitivity are shown in Table 5.

TABLE 5 Sensitivity Evaluation ATCC# Copies Number Number or per of ofReference Reac- Reac- Repli- Posi- Posi- Organism # tion tions catestives tivity L. grayi 19120 1E5 20 1 0  0% L. innocua 33090 1E5 20 1 20100% L. ivanovii F4081 1E5 20 1 20 100% L. murrayi F4076 1E5 20 1 0  0%L. seeligeri F4088 1E5 20 1 20 100% L. welshimeri F4082 1E5 20 1 20 100%L. 35152 1E5 40 1 40 100% monocytogenes* No Target* NA 0 16 1 0  0%*Total of all runs

Initial testing for Sensitivity was considered incomplete since the L.grayi and L. murrayi were not detected. All other species tested passedyielding a 100% positivity rate. No false positives were observed. L.grayi and L. murrayi are genotypically identical in the region ofamplification. They are also genotypically different from the otherspecies tested by only a one base mismatch in the specific bindingregion of the T7 provider oligo. Therefore, a redesign was necessary(see Example 5, below).

Specificity

Challenge organisms were tested at 1E5 copies per reaction(approximately 100 CFU) using the KINGFISHER 96 instrument for targetcapture and the BMG reader for detection. Twenty reactions of allchallenge organisms and the negative controls were tested in each platealong with 8 reactions of the positive control. From each reaction one30 uL replicate was amplified. L. monocytogenes, ATCC 35152, was used asa positive control at 1E5 copies per reaction and lysis solution used asa negative control. The positive criterion was 3000 RFU.

A criteria of less than or equal to 10 positives out of 200 reactions (1out of 20) would meet the goal of ≦5% false positivity rate. Thedispersion of any false positives across the 10 organisms was to beconsidered. Organisms with clustered false positivity (≧4) will bere-tested and further investigated. The results of Specificityevaluation are summarized in Table 6.

TABLE 6 Specificity Evaluation Copies Number Number per of of Reac-Reac- Repli- Posi- Posi- Organism ATCC# tion tions cates tives tivity E.rhusiopathiae 19414 1E5 20 1 0  0% B. thermosphacta 11509 1E5 20 1 0  0%E. cloacae 29941 1E5 20 1 0  0% C. freundii 33128 1E5 20 1 0  0% S.flexneri 12022 1E5 20 1 0  0% P. mirabilis 29906 1E5 20 1 0  0% E.faecalis 33186 1E5 20 1 0  0% E. coli 10798 1E5 20 1 0  0% C. jejuni33560 1E5 20 1 0  0% S. enteritidis 10376 1E5 20 1 0  0% L.monocytogenes 35152 1E5 32 1 32 100% (positive)* Negative* NA 0 80 1 0 0% *Total of all runs

Specificity evaluation showed 0% positivity against any of the challengeorganisms tested and 100% positivity with the positive control.

Interference

L. monocytogenes, ATCC 35152, was used as the baseline target at 1E5copies per reaction (approximately 100 CFU). Challenge organisms werespiked into the samples at a concentration of 0 (lysis solution only) or1E7 copies (approximately 10,000 CFU). Assays were performed using theKINGFISHER 96 processor and the BMG microplate reader. Twelve reactionsof all conditions were tested. From each reaction, one 30 uL replicatewas amplified. The positive criterion used was 3000 RFU.

Results were to report the reproducibility of positivity in the presenceof the nearest neighbor organisms. The dispersion of interference acrossthe organisms tested was to be considered. Organisms exhibitinginterference were to be retested and investigated further. The resultsof Interference evaluation are summarized in Table 7.

TABLE 7 Interference Evaluation Copies Number Number per of of Reac-Reac- Repli- Posi- Posi- Organism ATCC# tion tions cates tives tivity E.rhusiopathiae 19414 1E7 12 1 12 100% B. thermosphacta 11509 1E7 12 1 12100% E. cloacae 29941 1E7 12 1 12 100% C. freundii 33128 1E7 12 1 12100% S. flexneri 12022 1E7 12 1 12 100% P. mirabilis 29906 1E7 12 1 12100% E. faecalis 33186 1E7 12 1 12 100% E. coli 10798 1E7 12 1 12 100%C. jejuni 33560 1E7 12 1 12 100% S. enteritidis 10376 1E7 12 1 12 100%L. monocytogenes 35152 1E5 24 1 24 100% (positive)* Negative* NA 0 24 10  0% *Total of all runs

Interference evaluation showed 100% listeria positivity in the presenceof all challenge samples and 100% positivity with the positive control.

Example 5 Redesign of T7 Provider

Additional evaluation of set #3 (Table 3) revealed that theoligonucleotide set did not detect L. grayi and L. murrayi once targetcapture and the internal control were integrated into the system. Anadditional T7 provider was designed and evaluated. The goal of thedesign was to overcome the detrimental effects of the mismatch observedin the L. grayi and L. murrayi species tested while not affecting thedetection of other Listeria species/strains. The method of evaluatingthe redesigned oligo was performed in two sequential experiments. First,the redesigned T7 provider oligo was compared to the control T7 providerusing L. grayi and L. monocytogenes. Each T7 provider was tested byitself as well as together in an equal-molar mixture. Once it wasdetermined that the redesigned T7 provider allowed detection of the L.grayi species without loss of detection of the L. monocytogenes,Sensitivity testing was repeated in its entirety. Specificity andInterference evaluation were performed for only the more geneticallyrelated organisms, E. rhusiopathiae and B. thermosphacta.

Experiment 1: Amplification reagent containing either the control T7Provider (SEQ ID NO:13) or the redesigned T7 Provider (SEQ ID NO:14) wasformulated. In addition, a third amplification reagent was formulatedthat contained both T7 providers in an equal-molar ratio. L.monocytogenes (ATCC 35152) and L. grayi (ATCC 19120) were tested witheach of the three amplification reagents at levels of 0, 1E3, 1E4, and1E5 copies per reaction in replicates of 4. All samples were evaluatedbased on a positive criterion of 3000 RFU.

Experiment 2—Sensitivity, Specificity, and Interference Evaluation:Based on Experiment 1, amplification reagent containing an equal-molarratio of control T7 provider and redesigned T7 provider was formulated.Evaluation of the Sensitivity was the same as that performed for T7provider (SEQ ID NO: 13). For Specificity testing, E. rhusiopathiae(ATCC 19414) and B. thermosphacta (ATCC11509) were tested at 1E5 copiesper reaction (approximately 100 CFU). Likewise, the same challengeorganisms at 1E7 copies per reaction combined with L. monocytogenes(ATCC 35152) at 1E5 copies per reaction were tested to determine anychanges in Interference due to the addition of the redesigned T7provider oligo. Samples evaluated for Sensitivity were performed inreplicate reactions of 20, whereas the samples evaluated for Specificityand Interference were performed in replicate reactions of 12. From eachreaction, one 30 μL replicate was amplified. All samples were evaluatedbased on a positive criterion of 3000 RFU.

Results

Experiment 1: The sensitivity results for the T7-Provider redesign areshown in Table 8 and Table 9. Table 9 shows that the oligonucleotide Set3 with Control T7 Provider (SEQ ID NO:13) showed sensitivity fordetecting L. monocytogenes nucleic acid at 10³ copies per reaction, butrequired 10⁵ copies per reaction of L. grayi nucleic acid to score apositive reaction. Substituting the Redesigned T7 Provider (SEQ IDNO:14) for the Control T7 Provider enhanced sensitivity for detecting L.grayi nucleic acid and a mixture of Control and Redesigned T7 Providersgave even better sensitivity.

TABLE 8 L. grayi (ATCC 19120) Copies Number Number per of ofAmplification Reac- Reac- Repli- Posi- Posi- Organism Reagent tion tionscates tives tivity L. grayi Control T7 0 4 1 0  0% Provider 1E3 4 1 0 0% (SEQ ID 1E4 4 1 0  0% NO: 13) 1E5 4 1  4* 100% Redesigned 0 4 1 0 0% T7-Provider 1E3 4 1 3  75% (SEQ ID 1E4 4 1 4 100% NO: 14) 1E5 4 1 4100% Mixed 0 4 1 0  0% 1E3 4 1 4 100% 1E4 4 1 4 100% 1E5 4 1 4 100%*These were very low positives.

TABLE 9 L. monocytogenes (ATCC 35152) Copies Number Number per of ofAmplification Reac- Reac- Repli- Posi- Posi- Organism Reagent tion tionscates tives tivity L. Reactions 0 4 1 0  0% monocytogenes Control T7 1E34 1 4 100% Provider 1E4 4 1 4 100% (SEQ ID 1E5 4 1 4 100% NO: 13)Redesigned 0 4 1 0  0% T7-Provider 1E3 4 1 4 100% (SEQ ID 1E4 4 1 4 100%NO: 14) 1E5 4 1 4 100% Mixed 0 4 1 0  0% 1E3 4 1 4 100% 1E4 4 1 4 100%1E5 4 1 4 100%

Experiment 2: The results of Experiment 2, with the mixture of T7Providers from Experiment 1, for Sensitivity, Specificity, andInterference Evaluation are summarized in Table 10, Table 11, and Table12, respectively.

TABLE 10 Sensitivity Evaluation ATCC# Copies Number Number or per of ofReference Reac- Reac- Repli- Posi- Posi- Organism # tion tions catestives tivity L. grayi 19120 1E5 20 1 20 100% L. innocua 33090 1E5 20 120 100% L. ivanovii F4081 1E5 20 1 20 100% L. murrayi F4076 1E5 20 1 20100% L. seeligeri F4088 1E5 20 1 20 100% L. welshimeri F4082 1E5 20 1 20100% L. 35152 1E5 40 1 40 100% monocytogenes* No Target* NA 0 16 1 0  0%*Total of all runs

TABLE 11 Specificity Evaluation Copies Number Number per of of Reac-Reac- Repli- Posi- Posi- Organism ATCC# tion tions cates tives tivity E.rhusiopathiae 19414 1E5 12 1 0  0% B. thermosphacta 11509 1E5 12 1 0  0%L. monocytogenes 35152 1E5 12 1 12 100% (positive) Negative NA 0 12 1 0 0%

TABLE 12 Interference Evaluation Copies Number Number per of of Reac-Reac- Repli- Posi- Posi- Organism ATCC# tion tions cates tives tivity E.rhusiopathiae 19414 1E7 12 1 12 100% B. thermosphacta 11509 1E7 12 1 12100% L. monocytogenes 35152 1E7 12 1 12 100% (positive) Negative NA 0 121 0  0%

Evaluation of Sensitivity (Table 10) demonstrated that with a mixture ofT7 provider oligos, the L. grayi and L. murrayi that had been missedearlier, were detected with 100% positivity. There was no loss inspecificity or increase in interference to the two closest related(genetically) challenge organisms.

These results indicate that the detection of Listeria (all species) canbe achieved by the compositions and methods even in the presence ofclosely related organisms, based upon the characteristics of thereal-time TMA data (e.g., the size and shape of RFU curves generatedfrom the real-time TMA reactions).

Example 6 Alternative Regions

Amplification and detection oligonucleotides targeting the nucleotidebase region corresponding to bp 1180-1370 of E. coli (accession no.J01859) reference rRNA, hereinafter the “1275 region” (FIG. 3), wereprepared for evaluation. These oligonucleotides were designed to becomplementary or homologous to Listeria monocytogenes rRNA with as muchhomology to the other Listeria species in this region.

Table 13 presents sequences of T7 Provider oligonucleotides, Primeroligonucleotides, and other ancillary oligonucleotides (e.g., Blocker,Torch, and Target Capture oligonucleotides) that were designed for the1275 region by the invention.

TABLE 13 Examples of Preferred Oligonucleotides16S 1275 Region Sequences Use SEQ ID NO: 1275 Region SequenceTarget Capture SEQ ID NO: 37 gguguuacaaacucucguggugugacgTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA Blocker SEQ ID NO: 38 gaugauuugacgucauccccaccu-XBlocker SEQ ID NO: 39 gcaugaugauuugacgucauc-X Blocker SEQ ID NO: 40cauaaggggcaugaugauuugacg-X Blocker SEQ ID NO: 41cccaggucauaaggggcaugaug-X Blocker SEQ ID NO: 42 uguagcccaggucauaagggg-XBlocker SEQ ID NO: 43 acguguguagcccaggucauaag-X Blocker SEQ ID NO: 44auccauuguagcacguguguagcc-X T7 Provider SEQ ID NO: 45AATTTAATACGACTCACTATAGGGAGACTTATGA CCTGGGCTACACACGTGCTACAATGG-XT7 Provider SEQ ID NO: 46 AATTTAATACGACTCACTATAGGGAGAATCATCATGCCCCTTATGACCTGGGCTACA-X T7 Provider SEQ ID NO: 47AATTTAATACGACTCACTATAGGGAGAATCATCAT GCCCCTTATGACCTGGGCTACACACG-XT7 Provider SEQ ID NO: 48 AATTTAATACGACTCACTATAGGGAGACATGCCCCTTATGACCTGGGCTACACACGTGC-X T7 Provider SEQ ID NO: 49AATTTAATACGACTCACTATAGGGAGACATGCCC CTTATGACCTGGGCTACACACGTGCTA-XT7 Provider SEQ ID NO: 50 AATTTAATACGACTCACTATAGGGAGACTGGGCTACACACGTGCTACAATGGATAGT-X T7 Provider SEQ ID NO: 51AATTTAATACGACTCACTATAGGGAGACACACGT GCTACAATGGATAGTACAAAGGG-X T7 ProviderSEQ ID NO: 52 AATTTAATACGACTCACTATAGGGAGACTACACACGTGCTACAATGGATAGTACAAA-X T7 Provider SEQ ID NO: 53AATTTAATACGACTCACTATAGGGAGACTACACA CGTGCTACAATGGATAGTACAAAG-XT7 Provider SEQ ID NO: 54 AATTTAATACGACTCACTATAGGGAGACTACACACGTGCTACAATGGATAGTACAAAGG-X T7 Provider SEQ ID NO: 55AATTTAATACGACTCACTATAGGGAGACTACACA CGTGCTACAATGGATAGTACAAAGGG-XT7 Provider SEQ ID NO: 56 AATTTAATACGACTCACTATAGGGAGACTACACACGTGCTACAATGGATAGTACAAAGGGT-X T7 Provider SEQ ID NO: 57AATTTAATACGACTCACTATAGGGAGACACGTGC TACAATGGATAGTACAAAGGGTCGCG-XT7 Provider SEQ ID NO: 58 AATTTAATACGACTCACTATAGGGAGATGGATAGTACAAAGGGTCGCGGAAGCGCGAG-X Primer SEQ ID NO: 59ggcgAGTTGCAGCCTACAATCCGAACUG Primer SEQ ID NO: 60ggcgAGTTGCAGCCTACAATCCGAA Primer SEQ ID NO: 61ggcuTCATGTAGGCGAGTTGCAGCCTACA Primer SEQ ID NO: 62ggcuTCATGTAGGCGAGTTGCAGC Primer SEQ ID NO: 63cgauTCCGGCTTCATGTAGGCGAGTTGCAGC Primer SEQ ID NO: 64cuagCGAUTCCGGCTTCATGTAGGCGAGTTG Primer SEQ ID NO: 65ccacGATTACTAGCGATTCCGGCTTCATGTA Primer SEQ ID NO: 66gaucCACGATTACTAGCGATTCCGGCTT Primer SEQ ID NO: 67guggCATGCTGATCCACGATTACTAGCGA Primer SEQ ID NO: 68ggcaTGCTGATCCACGATTACTAGC Primer SEQ ID NO: 69 caccGTGGCATGCTGATCCACGATTTorch SEQ ID NO: 70 gcuccaccucgcgcuuggagc Torch SEQ ID NO: 71cugggauuagcuccaccucgcgcucccag Torch SEQ ID NO: 72cugggauuagcuccaccuccccag Torch SEQ ID NO: 73 ggagcuaaucccauaaaacuagcuccTorch SEQ ID NO: 74 cggaguaguuuuaugggauuagcuccg Torch SEQ ID NO: 75cggaggaauaguuuuaugggauuagcuccg Torch SEQ ID NO: 76cugagaauaguuuuaugggauuagcucccucag Torch SEQ ID NO: 77cgagaauaguuuuaugggauuagcuccucg Torch SEQ ID NO: 78cuaguuuuaugggauuagcuag Torch SEQ ID NO: 79 cgagaauaguuuuaugggauuagcucgTorch SEQ ID NO: 80 cugagaauaguuuuaugggauuagcucag Torch SEQ ID NO: 81cgaacugagaauaguuuuaugggauuagguucg Torch SEQ ID NO: 82ccgaacugagaauaguguucgg “X” = optional blocking moiety (e.g., reverse3′-5′-C); lower case n = 2′-O-methyl ribose; upper case N = deoxyribose;5′-fluorescein (“F”) fluorophore and 3′-dabsyl (“D”) quencher moietieswere attached to the torch oligonucleotides

Screening was performed the same as the screening for the 450 region ofthe 16S rRNA. L. grayi was chosen for the screening. Sequencealignments, see FIG. 3, showed that L. grayi, L. innocua, L. seeligeri,L. ivanovii and L. weishimeri have similar homologies and mismatchescompared to the L. monocytogenes sequence in the 1275 region, so it wasthought that detection of L. grayi would be representative of theability to detect nucleic acids from the Listeria species of interest.All screening was performed at 0 and 1E5 copies/reaction and utilizedthe BMG reader. The oligonucleotide sets in Table 14 showed the bestpotential to pick up the Listeria genus based on the TTime and thefluorescence signal for L. grayi.

TABLE 14 Listeria Oligonucleotide Sets 1275 Region OligonucleotideCombination SEQ ID NOs: RFU of Provider:Blocker:Primer:Torch TTime Range55:42:59:72 18.74 36,332 55:42:65:72 17.20 33,396 57:43:64:72 17.6036,675 47:38:64:77 18.99 32,468 55:42:65:77 17.00 41,038 49:39:59:7819.90 36,691 49:39:64:78 17.80 36,635 50:41:64:78 18.70 32,81652:42:68:78 18.27 34,443 55:42:63:78 19.55 34,649 55:42:64:78 15.1034,984 55:42:65:78 16.30 33,144 55:42:68:78 15.62 31,009 57:43:64:7816.72 40,238 57:43:65:78 16.95 30,116 45:40:64:79 19.90 52,74447:38:68:79 16.60 39,929 45:40:65:79 18.25 46,165 50:41:66:79 18.6541,209 52:42:65:79 19.91 40,711 55:42:65:79 17.29 42,281 55:42:67:7919.29 37,119 52:42:64:80 18.98 42,005 52:42:65:80 18.74 31,54047:38:64:81 18.60 34,542 57:43:67:81 18.67 30,890

In addition, some of the oligonucleotides in Table 13 were redesigned tobe more tolerant to the mismatches present in the sequence among theListeria subspecies and are shown in Table 15.

TABLE 15 16S 1275 Region Redesigned Sequences SEQ ID Use NO: 1275 Region Sequence T7 SEQ ID AATTTAATACGACTCACTATAGGGAGACTACACProvider NO: 83 ACGTGCTACAATGGATACTACAAA-X T7 SEQ IDAATTTAATACGACTCACTATAGGGAGACTACAC Provider NO: 84ACGTGCTACAATGGCTAGTACAAA-X T7 SEQ ID AATTTAATACGACTCACTATAGGGAGACACGTGProvider NO: 85 CTACAATGGATACTACAAAGGGTCGCG-X T7 SEQ IDAATTTAATACGACTCACTATAGGGAGACACGTG Provider NO: 86CTACAATGGCTAGTACAAAGGGTCGCG-X Primer SEQ ID GAGAATAGTTTTATGGGATTA NO: 87Primer SEQ ID GAGAATAGTTTTATGGGATCA NO: 88 Primer SEQ IDGAGAATAGTTTTATGCGATTA NO: 89 Primer SEQ ID GAGAATACTTTTATGGGATTA NO: 90Primer SEQ ID CTGAGAATAGTTTTATGGGATTA NO: 91 Primer SEQ IDCTGAGAATAGTTTTATGGGATCA NO: 92 Primer SEQ ID CTGAGAATAGTTTTATGCGATTANO: 93 Primer SEQ ID CTGAGAATACTTTTATGGGATTA NO: 94 Primer SEQ IDCCGAACTGAGAATAGTTTTATGGGATTA NO: 95 Primer SEQ IDccgaaCTGAGAATAGTITTATGGGATTA NO: 96 Primer SEQ IDCCGAACTGAGAATAGTTTTATGGGATCA NO: 97 Primer SEQ IDccgaaCTGAGAATAGTITTATGGGATCA NO: 98 Primer SEQ IDCCGAACTGAGAATAGTTTTATGCGATTA NO: 99 Primer SEQ IDccgaaCTGAGAATAGTITTATGCGATTA NO: 100 Primer SEQ IDCCGAACTGAGAATACTTTTATGGGATTA NO: 101 Primer SEQ IDccgaaCTGAGAATACTITTATGGGATTA NO: 102 “X” = optional blocking moiety(e.g., reverse 3′-5′-C); lower case n = 2′-O-methyl ribose; upper case N= deoxyribose; 5′-fluorescein (“F”) fluorophore and 3′-dabsyl (“D”)quencher moieties were attached to the torch oligonucleotides

Example 7 Food Testing of Spiked Ground Beef and Ice Cream

A small food study was performed using the 450 region Set #3 and TCO 2(see Examples 2 & 3) oligonucleotides to test ice cream and ground beefas food matrices. The study was conducted in two phases. The first was apre-study where the dilutions and CFU timing approximated. The secondphase of the study evaluated food with spiked Listeria. The samples werespiked with 10-20 CFU/25 g of food. The CFU count for the spiking wasbased on a McFarland 1. Specimens were sampled at 0, 4, 6, 8, 10, and 24hours. For each point in the time course, the sample was plated for CFUcounts, processed for storage and tested with the Listeria real-Time TMAassay.

The various steps followed in this study are described below. AMcFarland 1 of Listeria was made. CFU count confirmation in TSA plates(made dilution to 1E+6 in sterile PBS) was performed. Twenty-five gramsof food was weighed and aseptically placed into a STOMACHER bag. TwentyCFU were inoculated directly to 225 mL of Demi-Frazer. The spiked mediawas poured into the food-containing STOMACHER bag and processed for 2minutes at 200 rpm. The sample was incubated at 30° C. A 1-mL aliquotwas removed (1 aliquot for use in plate count) at times 0, 4, 6, 8, 10,and 24 hours. The sample was plated for CFU counts on selective agar,MOX plates at 3 dilutions, 1 plate/dilution and incubated at 35° C. Theremaining five aliquots sampled during a 24-hour period were spun at12,000×g for 30 seconds. The supernatant was removed and 500 μL of a 50mM succinate buffer (0.6 M LiCl, 1% LiLS, pH 4.8) was added to thepellet which was then vortexed vigorously for 20 seconds. The sample washeated at >90° C. at least 15 minutes. It was then spun at 12,000×g for1 minute. The supernatant was transferred to a new labeled tube. Sampleswere frozen at −70° C. Food controls included: 2 positive and 2 negativefor ground beef, 2 positive and 2 negative for plain vanilla ice cream,and 2 positive and 1 negative for the media only pure system.

Pre-Study/CFU Timing:

In the absence of food, spiked Listeria (˜15 CFU starting inoculum) grewto ˜270 CFU/ml after 6 h in broth.

Culture Results:

Using an inoculum of around 24 CFU, the spiked Listeria in ground beefgrew to around 15 CFU/mL after 8 hours of incubation in Demi-Frazerbroth. In spiked ice cream, 70 CFU/mL were observed after 10 hours ofincubation in Demi-Frazer broth. The unspiked ground beef wascontaminated with Listeria spp. or other organisms showing similarcultural characteristics as Listeria and had over 6E+4 CFU/mL after 24hours of incubation. The spiked media without any food sample had around10 CFU/mL after 10 hours of incubation. By 24 hours, all spiked samplesand unspiked ground beef sample in Demi-Frazer broth had >2.8E+4 CFU/mL.The unspiked ice cream and negative media control did not show anyListeria growth even after 24 hours incubation in Demi-Frazer broth. CFUcounts are shown in Table 16.

TABLE 16 CFU counts at various time points for food spiked with Listeriamonocytogenes GP803 (24 CFU inoculum) CFU Counts (CFU/mL) SamplingGround Beef Ice Cream Media Only Time (h) 0 0 0 0 0 0 0 0 0 0 0 0 0 4 00 0 0 0 0 6 0 0 0 0 0 0 8 15 0 0 0 0 0 10 40 0 70 0 10 024 >4.0E+5 >6.0E+4 >1.0E+5 0 >2.8E+4 0

Listeria Real-Time Amplification Results:

Real-Time amplification results are summarized below in Table 17.

TABLE 17 Real-Time Amplification Results Time Ice cream Ice cream MediaCtrl Media Ctrl (hours) Meat Spiked Meat Unspiked Spiked Unspiked SpikedUnspiked 0 Negative Negative Negative Negative Negative Negative 4Negative Negative Negative Negative Negative Negative 6 NegativeNegative Negative Negative Negative Negative 8 Negative NegativeNegative Negative Negative Negative 10 Not tested Not tested Not testedNot tested Not tested Not tested 24 Positive Low Positive PositiveNegative Positive Negative

Listeria was not detected in any samples until the 24-hour time point.The overall sensitivity of the assay is considered to be between 1E3 and1E4 copies/reaction (under ideal amplification conditions). Themagnitude of interference from the food and media matrices that maycause lower sensitivity than culture is unknown at this time.

SUMMARY

Amplification and detection oligonucleotides targeting the 450 and 1275regions of Listeria nucleic acid, corresponding to E. coli 16S rRNAnucleotide base positions at about 350-505 and about 1180-1370,respectively, were designed and synthesized for evaluation.

The 450 region Listeria assay was 100% sensitive to all 7 Listeriaspecies at 1E+5 copies/reaction (˜100 CFU). The Listeria assay was 100%specific against 10 non-Listeria organisms and Brochothrix andErysipelothrix. The limit of detection was 1-10 CFU. The rapid real-timeTMA assay can be run in less than four hours, reducing the time neededfor testing in food and manufacturing facilities from days to hours.

Oligonucleotides targeting the 1275 region also showed promising resultsas an alternative to those targeting the 450 region.

Increased assay sensitivity might be achieved by the use of additionalamplification and/or detection oligonucleotides designed to amplify anddetect the 450 region by itself or to amplify and detect the 450 as wellas the 1275 regions of Listeria.

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other physical and electronic documents.

The methods illustratively described herein may suitably be practiced inthe absence of any element or elements, limitation or limitations, notspecifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof. It is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the invention embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the methods. This includes the genericdescription of the methods with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the methods are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

1-30. (canceled)
 31. A method for detecting Listeria in a sample, themethod comprising: performing a nucleic acid amplification assay using aset of oligonucleotides comprising a T7 provider oligonucleotide and aprimer oligonucleotide, wherein the T7 provider oligonucleotide and theprimer oligonucleotide target sequences in a region of Listeria nucleicacid corresponding to SEQ ID NO:113, and wherein the T7 provideroligonucleotide and the primer oligonucleotide used in saidamplification assay target opposite strands of the Listeria nucleic acidsequence to be amplified, and detecting the presence or absence ofamplification product, wherein the presence of the amplification productindicates the presence of Listeria in the sample.
 32. The method ofclaim 31, wherein the detecting step utilizes a detectionoligonucleotide that specifically hybridizes to the amplificationproduct.
 33. The method of claim 32, wherein the detectionoligonucleotide is a molecular torch.
 34. The method of claim 33,wherein the T7 provider oligonucleotide has a sequence consisting of SEQID NO:55, the primer oligonucleotide has a sequence consisting of SEQ IDNO:65, and the molecular torch has a sequence consisting of SEQ IDNO:72, SEQ ID NO:77, SEQ ID NO:78, or SEQ ID NO:79.
 35. The method ofclaim 34, wherein the molecular torch has the sequence consisting of SEQID NO:72.
 36. The method of claim 34, wherein the molecular torch hasthe sequence consisting of SEQ ID NO:77.
 37. The method of claim 34,wherein the molecular torch has the sequence consisting of SEQ ID NO:78.38. The method of claim 34, wherein the molecular torch has the sequenceconsisting of SEQ ID NO:79.
 39. The method of claim 33, wherein the T7provider oligonucleotide has a sequence consisting of SEQ ID NO:55, theprimer oligonucleotide has a sequence consisting of SEQ ID NO:64, SEQ IDNO:65, or SEQ ID NO:68, and the molecular torch has a sequenceconsisting of SEQ ID NO:78.
 40. The method of claim 39, wherein theprimer oligonucleotide has the sequence consisting of SEQ ID NO:64. 41.The method of claim 39, wherein the primer oligonucleotide has thesequence consisting of SEQ ID NO:68.
 42. The method of claim 33, whereinthe T7 provider oligonucleotide has a sequence consisting of SEQ IDNO:57, the primer oligonucleotide has a sequence consisting of SEQ IDNO:64 or SEQ ID NO:65, and the molecular torch has a sequence consistingof SEQ ID NO:78.
 43. The method of claim 42, wherein the primeroligonucleotide has the sequence consisting of SEQ ID NO:64.
 44. Themethod of claim 42, wherein the primer oligonucleotide has the sequenceconsisting of SEQ ID NO:65.
 45. The method of claim 33, wherein the T7provider oligonucleotide has a sequence consisting of SEQ ID NO:57, theprimer oligonucleotide has a sequence consisting of SEQ ID NO:64, andthe molecular torch has a sequence consisting of SEQ ID NO:72 or SEQ IDNO:78.
 46. The method of claim 45, wherein the molecular torcholigonucleotide has the sequence consisting of SEQ ID NO:72.
 47. Themethod of claim 33, wherein the T7 provider oligonucleotide has asequence consisting of SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:55, or SEQID NO:57, the primer oligonucleotide has a sequence consisting of SEQ IDNO:64, and the molecular torch has a sequence consisting of SEQ IDNO:78.
 48. The method of claim 47, wherein the T7 provideroligonucleotide has the sequence consisting of SEQ ID NO:49.
 49. Themethod of claim 47, wherein the T7 provider oligonucleotide has thesequence consisting of SEQ ID NO:50.
 50. The method of claim 33, whereinthe T7 provider oligonucleotide has a sequence consisting of SEQ IDNO:47, the primer oligonucleotide has a sequence consisting of SEQ IDNO:68, and the molecular torch has a sequence consisting of SEQ IDNO:79.